Methods for variant detection

ABSTRACT

The invention can be used to provide a more efficient and less error-prone method of detecting variants in DNA, such as SNPs and indels. The invention also provides a method for performing inexpensive multiplex assays. The invention also provides methods for detection of DNA sequences altered after cleavage by a targetable endonuclease, such as the CRISPR Cas9 protein from the bacterium  Streptococcus pyogenes.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part of U.S. application Ser. No.15/361,280, filed Nov. 25, 2016, which claims the benefit of U.S.Provisional Application No. 62/339,317, filed May 20, 2016, and alsoclaims the benefit of U.S. Provisional Application No. 62/259,913, filedNov. 25, 2015, the disclosures of all of which are hereby incorporatedby reference in their entireties.

FIELD OF THE INVENTION

The invention can be used to provide a more efficient and lesserror-prone method of detecting variants in DNA, such as singlenucleotide polymorphisms (SNPs), multi-nucleotide polymorphisms (MNPs),and indels. The invention also provides a method for performinginexpensive multi-color assays, and provides methods for visualizingmultiple allele results in a two-dimensional plot. The invention alsoprovides methods for detection of DNA sequences altered after cleavageby a targetable endonuclease, such as the CRISPR Cas9 protein from thebacterium Streptococcus pyogenes.

BACKGROUND OF THE INVENTION

RNase H2-dependent PCR (rhPCR) (see U.S. Patent Application PublicationNo. US 2009/0325169 A1, incorporated by reference herein in itsentirety) and standard allele-specific PCR (ASPCR) can both be utilizedfor mutation detection. In ASPCR, the DNA polymerase performs themismatch discrimination by detection of a mismatch at or near the 3′ endof the primer. While ASPCR is sometimes successful in mismatchdetection, the discrimination can be limited, due to the low mismatchdetection ability of wild-type DNA polymerases.

In contrast with ASPCR, the mismatch sensitivity of the RNase H2 enzymein rhPCR allows for both sensitive detection of DNA mutations, andelimination of primer-dimer artifacts from the reaction. When attemptingto detect DNA mutations with rhPCR, however, placement of the mismatchwithin the primer is important. The nearer to the cleavable RNA themismatch is located, the more discrimination is observed from the RNaseH2 enzyme, and the greater the discrimination of the resulting rhPCRassay. Given the fact that most common wild-type DNA polymerases such asTaq often display low levels of mismatch detection, the polymerasecannot be solely relied upon to perform this discrimination after RNaseH2 cleavage. Coupled with the repeated interrogation desired from everycycle of standard rhPCR, placing the mismatch anywhere other thanimmediately opposite the RNA is undesirable when utilizing thesepolymerases.

There is thus a need for assays with improved mismatch sensitivity.

In addition, there is a need for improved methods for detection ofmutations altered after cleavage by targetable endonucleases, such asthe CRISPR Cas9 protein. A commonly used method to detect mutationsintroduced into genomic DNA following repair of dsDNA cleavage events isthe enzymatic mismatch cleavage assay (EMCA). EMCA assays cleave atsites where base mismatches are present in dsDNA. For EMCA detection ofthe mutations introduced into DNA following Cas9 cleavage and repair,genomic DNA from cells is harvested and the regions around the dsDNA cutsite is amplified by PCR using primers that flank the cut site.Typically 100-1000 base amplicons are used for this purpose. Followingcompletion of amplification, heteroduplexes are formed by heating thereaction products and allowing them to re-anneal, which leads toformation of homoduplex WT/WT, Mut/Mut or heteroduplex WT/Mut orMut1/Mut2 variants. The dsDNAs are then subjected to cleavage by amismatch endonuclease (such as T7EI, Surveyor, etc.). Heteroduplexes arecleaved and the presence of shorter fragments is detected by gelelectrophoresis, capillary electrophoresis, or any of a number ofmethods known to those of skill in the art. Although such an assay isfast and inexpensive, it often does not accurately reflect the changesthat are actually generated from the CRISPR mutagenesis process. If thesame mutation is introduced a large number of times, Mut/Mut homodimersform, which are not detected. Further, the mismatch endonuclease enzymesoften fail to cleave single-base events, leading to yet another class ofmutations that are undetected. Thus an EMCA assay will almost alwaysunderestimate the extent of genome editing that occurred after Cas9dsDNA cleavage and repair.

An alternative method of analysis involves large scale DNA sequencingusing “Next-Gen” sequencing (NGS) methods of the modified DNA, which ishighly accurate, but is slow and costly. Other methods are available toassess the mutation outcome following CRISPR/Cas9 cleavage and repair.For example, Sanger sequencing results can be analyzed using sequencetrace decomposition (“TIDE” analysis); fluorescent-labeledprimer-extension on an amplicon spanning the Cas9 cut site can be usedto map indels using Indel Detection by Amplicon Analysis (IDAA); or highresolution melt analysis (HRM) can be applied to PCR amplicons that spanthe Cas9 cut site. However, none of these methods approaches theaccuracy of NGS analysis, while all are more costly and slower toperform than EMCA methods. Thus, improved methods are needed to assessthe frequency of mutations that arise from genome editing experimentsthat are rapid and low cost.

BRIEF SUMMARY OF THE INVENTION

The disclosure provides assays making use of high discriminationpolymerase mutants or other high mismatch discrimination polymerases tocreate a new assay design that can utilize mismatches located 5′ of theRNA.

The invention can be used to provide a more efficient and lesserror-prone method of detecting mutations in DNA, such as SNPs andindels. The invention also provides a method for performing inexpensivemulti-color assays. The invention also provides methods for detection ofDNA sequences altered after cleavage by a targetable endonuclease, suchas the CRISPR Cas9 protein from the bacterium Streptococcus pyogenes.

These and other advantages of the invention, as well as additionalinventive features, will be apparent from the description of theinvention provided herein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a diagram showing two primer designs utilized in thisinvention. Part a) is a blocked-cleavable primer designed so that theSNP of interest is 5′ of the RNA base when hybridized to a template. TheRNase H2 cleaves, leaving a 3′ interrogating base, which is determinedto be either a match or a mismatch by the highly discriminative DNApolymerase. Thermal cycling allows for this process to continue. Part b)illustrates the RNase H2 cleavage and SNP detection are identical to a),but the primer also includes a 5′ “tail” domain that includes a bindingsite for a probe and a universal forward primer. After 1-10 cycles ofdiscrimination with the RNase H2 and the polymerase, the highlyconcentrated universal forward primer comes to dominate theamplification, degrading the probe when it amplifies. This cycle isrepeated 25-50×, generating the output signal. This primer design may bemultiplexed, allowing for one-tube multi-color assay designs.

FIGS. 2A and 2B are end-point fluorescence plots from the assaydescribed in Example 1. FAM and HEX fluorescence values are plotted ontothe X and Y axis. FIG. 2A is a “Universal” SNP assay for rs351855performed with WT Taq polymerase. FIG. 2B is a “Universal” SNP assay forrs351855 performed with mutant H784Q Taq polymerase, demonstratinggreatly enhanced discrimination between each of the allelic variants asobserved by the greater separation of the clusters in the mutant Taqcase. In both cases, the no template controls (NTCs) (squares) are nearthe (0,0) coordinates, as desired. Allele 1 samples are shown ascircles, allele 2 samples as diamonds, and heterozygotes as triangles.Each reaction was performed in triplicate.

FIGS. 3A and 3B are allelic discrimination plots with genotyping callsfor rs4655751. The reaction plate was cycled immediately after reactionsetup (A) or held at room temperature on the benchtop for 48 hours priorto cycling (B). Diamonds: no template controls (NTCs); squares: allele 1samples; circles: allele 2 samples; triangles: heterozygotes. Genotypesare tightly clustered and have good angle separation, indicatingexcellent allelic specificity. Each sample was assigned the correctgenotyping call, and no change in performance was observed over the 48hour hold period.

FIGS. 4A and 4B illustrate a side-by-side comparison of AllelicDiscrimination Plots of gene CCR2, rs1799865 from a TaqMan based assayversus rhPCR. Diamonds: no template controls (NTCs); squares: allele 1samples; circles: allele 2 samples; triangles: heterozygotes. The rhPCRGenotyping Assay (FIG. 4B) achieved higher fluorescence signal comparedto a traditional 5′-nuclease genotyping assay (FIG. 4A) while showingconcordant results.

FIGS. 5A and 5B are Allelic Discrimination plots of tri-allelic SNP,CYP2C8 (r572558195), using an rhPCR genotyping single tube multiplexassay on the QuantStudio™ 7 Flex platform (Thermo Fisher). In FIG. 5A,diamonds: no template controls (NTCs); squares: allele G (allele 1)samples; circles: allele A (allele 2) samples; triangles: heterozygotes.In FIG. 5B, diamonds: no template controls (NTCs); squares: allele G(allele 1) samples; circles: allele C (allele 3) samples; triangles:heterozygotes.

FIG. 6 shows the Tri-allelic Allelic Discrimination 360plot of CYP2C8rs72558195, using rhPCR genotyping assay with 3 allele-specific primersmultiplexed in a single reaction.

FIG. 7 is an allelic discrimination plot illustrating the ability of therhPCR assay to perform quantitative genotyping.

FIGS. 8A and 8B illustrate genotyping results and detection of alleliccopy number variation that is possible with the present invention. gDNAsamples were tested using varying copy numbers and varying referencegenotypes. In FIG. 8A, diamonds: no template controls (NTCs); squares:allele G samples; circles: allele C samples; and triangles:heterozygotes. The resulting data correlates with the test input.

FIG. 9 is a schematic representation of multiplex rhPCR.

FIG. 10 is the resulting tape station image indicating the effectivenessof the multiplex rhPCR methods in reducing primer dimers and increasingdesired amplicon yield.

FIG. 11 graphically represents the effectiveness of the rhPrimers in thepercent of mapped reads and on-target reads.

FIG. 12 shows placement of the RNA residue relative to the most commoncleavage site for the methods of the disclosure (such as in Example 10).The RNA is shown in lower case, while DNA residues are shown in uppercase. Cas9 cleavage site is shown with a line through both strands ofDNA. RN2=RNase H2 enzyme; pol=polymerase.

FIGS. 13A-13B show analysis of CRISPR mutations demonstrating that theresults obtained using the qPCR methods of the disclosure are moreaccurate than using the T7EI EMCA method. FIG. 13A: the percentage ofmutated templates is consistently underestimated by T7EI EMCA (emptysquares), when compared with NGS results (grey circles) on the samesamples. FIG. 13B: using the same samples from FIG. 13A, the percentageof mutations detected is seen to be much more accurately estimated bythe qPCR methods of the disclosure (empty squares) when compared withNGS results.

DETAILED DESCRIPTION OF THE INVENTION

The invention pertains to a methods of single-nucleotide polymorphism(SNP) discrimination utilizing blocked-cleavable rhPCR primers (see U.S.Patent Application Publication No. US 2009/0325169 A1, incorporated byreference herein in its entirety) and a DNA polymerase with high levelsof mismatch discrimination. In one embodiment, the mismatch is placed ata location other than opposite the RNA base. In these situations, themajority of the discrimination comes not from the RNase H2, but from thehigh discrimination polymerase. The use of blocked-cleavable primerswith RNase H2 acts to reduce or eliminate primer-dimers and provide someincreased amount of SNP or indel (insertion/deletion) discrimination(FIG. 1a ).

For the purposes of this invention, high discrimination is defined asany amount of discrimination over the average discrimination of WTThermus aquaticus (Taq) polymerase. Examples include KlenTaq® DNApolymerase (Wayne Barnes), and mutant polymerases described in U.S.Patent Application Publication No. US 2015/0191707 (incorporated byreference herein in its entirety) such as H784M, H784S, H784A and H784Qmutants.

In a further embodiment a universal detection sequence(s) is added tothe 5′-end of the blocked-cleavable primers. The detection sequenceincludes a binding site for a probe, and a binding site for a universalamplification primer. The primer binding site is positioned at or nearthe 5′-end of the final oligonucleotide and the probe binding site ispositioned internally between the universal primer site and theSNP-detection primer domain. Use of more than one such chimeric probe ina detection reaction wherein distinct probe binding sites are employedallows primers to be multiplexed and further allows for multiple colordetection of SNPs or other genomic features. Blocked-cleavable rhPCRprimers reduce or eliminate primer-dimers. Primer-dimers are a majorproblem for use of “universal” primer designs in SNP detection assays,and that limits their utility (FIG. 1b ). Combining a universalamplification/detection domain with a SNP primer domain inblocked-cleavable primer format overcomes this difficulty.

Previously, the best preferred embodiment for rhPCR SNP discriminationemployed blocked-cleavable primers having the mismatch (SNP site)positioned opposite the single RNA base (cleavage site). While thisworks for many SNP targets, there are base match/mismatch pairings wheresufficient discrimination is not obtained for robust base calling.Moreover, due to the high level of differential SNP discriminationobserved with rhPCR, end-point detection can be difficult, especiallywith heterozygous target DNAs. In the proposed method, the RNA base isidentical in both discriminating primers, eliminating this issue.

In one embodiment of the invention, the method involves the use ofblocked-cleavable primers wherein the mismatch is placed 1-2 bases 5′ ofthe RNA. In a further embodiment, the method involves the use ofblocked-cleavable primers with three or more DNA bases 3′ of an RNAresidue, and the primers are designed such that the mismatch is placedimmediately 5′ of the RNA.

Following cleavage by RNase H2, the remaining primer has a DNA residuepositioned at the 3′-end exactly at the SNP site, effectively creatingan ASPCR primer. In this configuration, a high-specificity DNApolymerase can discriminate between match and mismatch with the templatestrand (FIGS. 1a and b ). Native DNA polymerases, such as Taq DNApolymerase, will show some level of discrimination in this primerconfiguration, and if the level of discrimination achieved is notsufficient for robust SNP calling in a high throughput assay format thenthe use of polymerases with improved template discrimination can beused. In one embodiment, mutant DNA polymerases, such as those disclosedin U.S. Patent Application Publication No. US 2015/0191707 (incorporatedby reference herein in its entirety) or any other polymerase designed oroptimized to improve template discrimination can be used. When usingpolymerases with increased mismatch discrimination, the final level ofmatch/mismatch discrimination achieved will be additive withcontributions from both the ASPCR primer polymerase interaction and fromthe rhPCR primer/RNaseH2 interaction. Further, the use ofblocked-cleavable primers reduces risk of primer-dimer formation, whichproduces false-positive signals, making the overall reaction more robustand having higher sensitivity and higher specificity. The relativecontributions of each component of the assay may vary with use ofdifferent polymerases, different blocking groups on the 3′-end of theprimer and different RNase H2 enzymes.

In another embodiment, the invention may utilize a “tail” domain addedto the 5′ end of the primer, containing a universal forward primerbinding site sequence and optionally a universal probe sequence. Thistail would not be complementary to the template of interest, and when aprobe is used, the tail would allow for inexpensive fluorescent signaldetection, which could be multiplexed to allow for multiple color signaldetection in qPCR (FIG. 1b ). In one embodiment, 1-10 cycles of initialcycling and discrimination occurs from both the RNase H2 and the DNApolymerase. After this initial pre-cycling, a highly concentrated andnon-discriminatory universal forward primer comes to dominate theamplification, degrading the probe and generating the fluorescent signalwhen the DNA amplifies. This cycle is repeated 25-50×, allowing forrobust detection. This assay design is prone to issues withprimer-dimers, and the presence of the blocked-cleavable domain in theprimers will suppress or eliminate these issues.

In another embodiment, a forward primer is optionally used with areverse primer, and a tail domain is added to the 5′ end of one or bothof a forward and reverse primer set. The tail domain comprises auniversal forward primer binding site. The primers can be used tohybridize and amplify a target such as a genomic sample of interest. Theprimers would add universal priming sites to the target, and furthercycles of amplification can be performed using universal primers thatcontain adapter sequences that enable further processing of the sample,such as the addition of P5/P7 flow cell binding sites and associatedindex or barcoding sequences useful in adapters for next-generationsequencing (see FIG. 9). In a further embodiment a high fidelitypolymerase is used, which will further lower the rate of basemisincorporation into the extended product and increase the accuracy ofthe methods of the invention.

As noted in U.S. Patent Application Publication No. US 2009/0325169(incorporated by reference herein in its entirety), RNase H2 can cleaveat positions containing one or more RNA bases, at 2′-modifiednucleosides such as 2′-fluoronucleosides. The primers can also containnuclease resistant linkages such as phosphorothioate,phosphorodithioate, or methylphosphonate.

Further aspects of the disclosure pertain to detection of DNA sequencesaltered after cleavage by a targetable endonuclease, such as the CRISPRCas9 protein from the bacterium Streptococcus pyogenes. This protein andsimilar ones have successfully been used for targeted genomicmodification in the well documented Clustered Regularly InterspacedShort Palindromic Repeats (CRISPR) system.

In a further embodiment, the tailed primers detailed above could be usedto detect editing events for genome editing technology. For example,CRISPR/Cas9 is a revolutionary strategy in genome editing that enablesgeneration of targeted, double-stranded breaks (DSBs) in genomic DNA.Methods to achieve DSBs by CRISPR/Cas9—a bacterial immune defense systemcomprised of an endonuclease that is targeted to double-stranded DNA bya guide RNA—are widely used in gene disruption, gene knockout, geneinsertion, etc. In mammalian cells, the endonuclease activity isfollowed by an endogenous repair process that leads to some frequency ofinsertions/deletions/substitutions in wild-type DNA at the target locuswhich gives the resultant genome editing.

RNase H-cleavable primers have been designed to flank edited loci inorder to 1) generate locus-specific amplicons with universal tails, and2) be subsequently amplified with indexed P5/P7 universal primers fornext-generation sequencing. In pilot experiments, this strategy resultedin reliable, locus-specific amplification which captures CRISPR/Cas9editing events in a high-throughput and reproducible manner. The keyfinding is that the overall targeted editing by this NGS-based methodwas determined to be 95%; whereas, previous enzymatic strategiessuggested overall editing from the same samples was approximately 55% atthe intended target site. Further, primers were designed to amplifyoff-target locations of genomic editing based on in silico predictionsby internal bioinformatics tools.

These assays would be pooled for amplification of a single genomic DNAsample in order to capture the on-target as well as >100 potential sitesfor off-target genome editing mediated by sequence homology to the guideRNA. The results from this experiment would allow for 1) identificationof CRISPR/Cas9 off-target sites and provide an assay for comparingstrategies to reduce those effects, 2) improved design of theCRISPR/Cas9 off-target prediction algorithm, and 3) improved design ofprimer sets.

Thus, in further aspects, the disclosure provides methods that employthe above-described universal rhPCR assay system to detect mutationsgenerated by a targetable endonuclease such as Cas9 or Cpf1. The rhPCRassays according to these aspects of the disclosure utilizes athermostable RNase H2 enzyme, and optionally a DNA polymerase withenhanced mismatch discrimination. The RNase H2 cleaves at the single RNAresidue only when the primer oligonucleotide is duplexed with a targetnucleic acid, which removes a 3′-blocking group and activates theprimer. The DNA polymerase uses the primer to initiate DNA synthesisand, in multiple cycles, supports PCR. Discrimination of mutations isachieved by the action of the RNase H2 or the combined action of boththe RNase H2 and the DNA polymerase, wherein the RNase H2 has reducedde-blocking activity when a mismatch is present and the DNA polymerasehas reduced priming/DNA synthesis activity when a mismatch is present.In one embodiment, the primers comprise multiple functional domainsincluding (from the 5′-end): a universal primer domain, a universalprobe binding domain, a target-specific primer domain, a single RNAresidue (cleavable linkage), a short 3′-extension domain, and a3′-blocking group that prevents the oligonucleotide from priming DNAsynthesis. Cleavage by RNase H2 removes the RNA residue, 3′-extensiondomain, and 3′-blocking group.

In some embodiments, a second assay is present in the reaction and runsas a 2-color multiplex, targeting the RNase P gene or some other controlgene. This second assay allows for normalization to an internal controlgene that was not targeted by the CRISPR genome editing reaction. Thiscontrol assay may be performed as either a standardthree-oligonucleotide 5′ nuclease assay, or as a second rhPCR-baseduniversal assay.

In another embodiment, the primers lack the universal 5′ domain, butstill retain the 3′ removable blocking group. In this alternativeembodiment, a standard 5′ nuclease fluorescence-quenched probe is placedbetween the forward and reverse primers. The probe is positioned withinthe amplicon such that it lies outside of any region that may be alteredby the genome editing event.

In each experiment, relative position of the discriminatory (i.e.,mutation interrogating) primer on the sequence is important. RNase H2cleaves 5′ of an RNA residue. Placement of the primer so that the RNAresidue binds two nucleotides after the most common cleavage site isimportant for recognition of the mutagenized samples. A diagram of thisprinciple is shown in FIG. 12. In the Wild-Type (WT) samples,amplification occurs normally, as neither the RNase H2 nor the DNApolymerase are hindered in their functions. If an insertion isintroduced to the sequence, a mismatch for both the RNase H2 and the DNApolymerase are produced, allowing two independent chances to distinguishmutant from WT. The same interrogation of the samples is achieved if adeletion is present—both the RNase H2 and the DNA polymerase detect themismatches generated (FIG. 12). This double level of interrogationallows for very precise quantification of the presence of mutated DNA ina heterogeneous sample.

Thus, in another aspect, the disclosure provides methods of detectingvariations in target DNA sequences that have been altered with a geneediting enzyme, the methods comprising: (a) providing a reaction mixturecomprising: (i) an oligonucleotide primer having a cleavage domainpositioned 5′ of a blocking group and 3′ of a position of variation, theblocking group linked at or near the end of the 3′-end of theoligonucleotide primer wherein the blocking group prevents primerextension and/or inhibits the primer from serving as a template for DNAsynthesis; (ii) a sample nucleic acid that may or may not have thetarget sequence, and where the target sequence may or may not have thevariation; (iii) a cleaving enzyme; and (iv) a polymerase; (b)hybridizing the primer to the target DNA sequence to form adouble-stranded substrate; (c) cleaving the hybridized primer, if theprimer is complementary at the variation, with the cleaving enzyme at apoint within or adjacent to the cleavage domain to remove the blockinggroup from the primer; and (d) extending the primer with the polymerase.

In some embodiments, the target DNA sequence has been treated with aCRISPR enzyme. In some embodiments, the target DNA sequence has beentreated with a Cas9 or Cpf1 enzyme. In some embodiments, the cleavingenzyme is a hot start cleaving enzyme which is thermostable and hasreduced activity at lower temperatures. In some embodiments, thecleaving enzyme is an RNase H2 enzyme. In some embodiments, the cleavingenzyme is Pyrococcus abyssi RNase H2 enzyme. In some embodiments, thecleaving enzyme is a chemically modified hot start cleaving enzyme whichis thermostable and has reduced activity at lower temperatures. In someembodiments, the hot start cleaving enzyme is a chemically modifiedPyrococcus abyssi RNase H2. In some embodiments, the cleaving enzyme isa hot start cleaving enzyme that is reversibly inactivated throughinteraction with an antibody at lower temperatures.

In some embodiments, the cleavage domain comprises at least one RNAbase, and the cleaving enzyme cleaves between the position complementaryto the variation and the RNA base. In some embodiments, the cleavagedomain comprises at least one RNA base located 3′ of the position ofvariation, and comprises one DNA base between the position of variationand the RNA base. In some embodiments, there are no DNA bases betweenthe position of variation and the RNA base. In other embodiments, theRNA base is located within the position of variation. In someembodiments, the cleavage domain comprises one or more 2′-modifiednucleosides, and the cleaving enzyme cleaves between the positioncomplementary to the variation and the one or more modified nucleosides.In some embodiments, the one or more modified nucleosides are2′-fluoronucleosides.

In some embodiments, the polymerase is a high-discrimination polymerase.In some embodiments, the polymerase is a mutant H784Q Taq polymerase. Insome embodiments, the mutant H784Q Taq polymerase is reversiblyinactivated via chemical, aptamer, or antibody modification. In someembodiments, the primer contains a 5′ tail sequence that comprises auniversal primer sequence and optionally a universal probe sequence,wherein the tail is non-complementary to the target DNA sequence.

In some embodiments, the methods of this aspect of the disclosurefurther comprise (e) detection of an internal control gene not targetedby the gene editing enzyme; and (f) normalization of the results ofsteps (a)-(d) to the results of step (e). In some embodiments, theinternal control gene not targeted by the gene editing enzyme is theRNase P gene. In some embodiments, the reaction mixture furthercomprises a control oligonucleotide primer specific for the internalcontrol gene not targeted by the gene editing enzyme, wherein thecontrol oligonucleotide primer comprises a cleavage domain positioned 5′of a blocking group and 3′ of a position of variation, the blockinggroup linked at or near the end of the 3′-end of the oligonucleotideprimer wherein the blocking group prevents primer extension and/orinhibits the primer from serving as a template for DNA synthesis. Insome embodiments, the internal control gene not targeted by the geneediting enzyme is detected using a three-oligonucleotide 5′ nucleaseassay.

In another aspect, the disclosure provides methods of target enrichmentcomprising: (a) providing a reaction mixture comprising: (i) a firstoligonucleotide primer having a tail domain that is not complementary toa target sequence, the tail domain comprising a first universal primersequence; a cleavage domain positioned 5′ of a blocking group and 3′ ofa position of variation, the blocking group linked at or near the end ofthe 3′-end of the first oligonucleotide primer wherein the blockinggroup prevents primer extension and/or inhibits the first primer fromserving as a template for DNA synthesis; (ii) a sample nucleic acid thathas been treated with a gene editing enzyme, which may or may not havethe target sequence; (iii) a cleaving enzyme; and (iv) a polymerase; (b)hybridizing the first primer to the target DNA sequence to form adouble-stranded substrate; (c) cleaving the hybridized first primer, ifthe first primer is complementary to the target, with the cleavingenzyme at a point within or adjacent to the cleavage domain to removethe blocking group from the first primer; and (d) extending the firstprimer with the polymerase.

In some embodiments, the target DNA sequence is a sample that has beentreated with a CRISPR enzyme. In some embodiments, the target DNAsequence is a sample that has been treated with a Cas9 or Cpf1 enzyme.In some embodiments, the methods further comprise a second primer inreverse orientation to support priming and extension of the first primerextension product. In some embodiments, the second primer furthercomprises a tail domain comprising a second universal primer sequence.In some embodiments, steps (b)-(d) are performed 1-10 times. In someembodiments, the methods further comprise removing unextended primersfrom the reaction and hybridizing universal primers to the extensionproduct to form a second extension product. In some embodiments, theuniversal primers further comprise tailed sequences for addition ofadapter sequences to the second extension product. In some embodiments,sequencing is performed on the second extension product to determine thesequence of the target.

In some embodiments, the cleaving enzyme is a hot start cleaving enzymewhich is thermostable and has reduced activity at lower temperatures. Insome embodiments, the cleaving enzyme is an RNase H2 enzyme. In someembodiments, the cleaving enzyme is Pyrococcus abyssi RNase H2 enzyme.In some embodiments, the cleaving enzyme is a chemically modified hotstart cleaving enzyme which is thermostable and has reduced activity atlower temperatures. In some embodiments, the hot start cleaving enzymeis a chemically modified Pyrococcus abyssi RNase H2. In someembodiments, the cleaving enzyme is a hot start cleaving enzyme that isreversibly inactivated through interaction with an antibody at lowertemperatures which is thermostable and has reduced activity at lowertemperatures. In some embodiments, the cleavage domain comprises atleast one RNA base, and the cleaving enzyme cleaves between the positioncomplementary to the variation and the RNA base.

In some embodiments, the cleavage domain comprises at least one RNA baselocated 3′ of the position of variation, and comprises one DNA basebetween the position of variation and the RNA base. In some embodiments,there are no DNA bases between the position of variation and the RNAbase. In other embodiments, the RNA base is located within the positionof variation. In some embodiments, the cleavage domain comprises one ormore 2′-modified nucleosides, and the cleaving enzyme cleaves betweenthe position complementary to the variation and the one or more modifiednucleosides. In some embodiments, the one or more modified nucleosidesare 2′-fluoronucleosides. In some embodiments, the polymerase is ahigh-discrimination polymerase. In some embodiments, the polymerase is amutant H784Q Taq polymerase. In some embodiments, the mutant H784Q Taqpolymerase is reversibly inactivated via chemical, aptamer or antibodymodification.

In another aspect, the disclosure provides blocked-cleavable primers forrhPCR, comprising: 5′-A-B-C-D-E-3′, wherein A is optional and is a tailextension that is not complementary to a target; B is a sequence domainthat is complementary to a target; C is a discrimination domain; D is acleavage domain that, when hybridized to the target, is cleavable byRNase H2, and which comprises an RNA base; and E is a blocking domainthat prevents extension of the primer.

In some embodiments, D is a cleavage domain that, when hybridized to thetarget, is cleavable by RNase H2, and which comprises an RNA base thatis: separated from the discrimination domain by one base position,within the discrimination domain, or adjacent to the discriminationdomain. In some embodiments, the RNA base is separated from thediscrimination domain by one base position. In some embodiments, the RNAbase is within the discrimination domain. In some embodiments, the RNAbase is adjacent to the discrimination domain. For example, when the RNAbase is adjacent to the discrimination domain, no intervening DNAresidue is present between the RNA base and the discrimination domain.

In some embodiments, D comprises 1-3 RNA bases. In some embodiments, thecleavage domain comprises one or more of the following moieties: a DNAresidue, an abasic residue, a modified nucleoside, or a modifiedphosphate internucleotide linkage. In some embodiments, a sequenceflanking the cleavage site contains one or more internucleoside linkagesresistant to nuclease cleavage. In some embodiments, the nucleaseresistant linkage is a phosphorothioate. In some embodiments, the 3′oxygen atom of at least one of the RNA residues is substituted with anamino group, thiol group, or a methylene group. In some embodiments, theblocking group is attached to the 3′-terminal nucleotide of the primer.In some embodiments, A is comprised of a region that is identical to auniversal forward primer and optionally a probe binding domain.

In some embodiments, the discrimination domain C does not comprise oroverlap with the cleavage domain D. In some other embodiments, thediscrimination domain C comprises the cleavage domain D. In some otherembodiments, the discrimination domain C overlaps with the cleavagedomain D.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art. In case of conflict, the present document, includingdefinitions, will control. Preferred methods and materials are describedbelow, although methods and materials similar or equivalent to thosedescribed herein can be used in practice or testing of the presentinvention.

“Complement” or “complementary” as used herein means a nucleic acid, andcan mean Watson-Crick (e.g., A-T/U and C-G) or Hoogsteen base pairingbetween nucleotides or nucleotide analogs of nucleic acid molecules.

“Fluorophore” or “fluorescent label” refers to compounds with afluorescent emission maximum between about 350 and 900 nm.

“Hybridization” as used herein, refers to the formation of a duplexstructure by two single-stranded nucleic acids due to complementary basepairing. Hybridization can occur between fully complementary nucleicacid strands or between “substantially complementary” nucleic acidstrands that contain minor regions of mismatch. “Identical” sequencesrefers to sequences of the exact same sequence or sequences similarenough to act in the same manner for the purpose of signal generation orhybridizing to complementary nucleic acid sequences. “Primer dimers”refers to the hybridization of two oligonucleotide primers. “Stringenthybridization conditions” as used herein means conditions under whichhybridization of fully complementary nucleic acid strands is stronglypreferred. Under stringent hybridization conditions, a first nucleicacid sequence (for example, a primer) will hybridize to a second nucleicacid sequence (for example, a target sequence), such as in a complexmixture of nucleic acids. Stringent conditions are sequence-dependentand will be different in different circumstances. Stringent conditionscan be selected to be about 5-10° C. lower than the thermal meltingpoint (™) for the specific sequence at a defined ionic strength pH. TheTm can be the temperature (under defined ionic strength, pH, and nucleicconcentration) at which 50% of an oligonucleotide complementary to atarget hybridize to the target sequence at equilibrium (as the targetsequences are present in excess, at Tm, 50% of the probes are occupiedat equilibrium). Stringent conditions can be those in which the saltconcentration is less than about 1.0 M sodium ion, such as about0.01-1.0 M sodium ion concentration (or other salts) at pH 7.0 to 8.3and the temperature is at least about 30° C. for short probes (e.g.,about 10-50 nucleotides) and at least about 60° C. for long probes(e.g., greater than about 50 nucleotides). Stringent conditions can alsobe achieved with the addition of destabilizing agents such as formamide.For selective or specific hybridization, a positive signal can be atleast 2 to 10 times background hybridization. Exemplary stringenthybridization conditions include the following: 50% formamide, 5×SSC,and 1% SDS, incubating at 42° C., or, 5×SSC, 1% SDS, incubating at 65°C., with wash in 0.2×SSC, and 0.1% SDS at 65° C.

The terms “nucleic acid,” “oligonucleotide,” or “polynucleotide,” asused herein, refer to at least two nucleotides covalently linkedtogether. The depiction of a single strand also defines the sequence ofthe complementary strand. Thus, a nucleic acid also encompasses thecomplementary strand of a depicted single strand. Many variants of anucleic acid can be used for the same purpose as a given nucleic acid.Thus, a nucleic acid also encompasses substantially identical nucleicacids and complements thereof. A single strand provides a probe that canhybridize to a target sequence under stringent hybridization conditions.Thus, a nucleic acid also encompasses a probe that hybridizes understringent hybridization conditions.

Nucleic acids can be single stranded or double stranded, or can containportions of both double stranded and single stranded sequences. Thenucleic acid can be DNA, both genomic and cDNA, RNA, or a hybrid, wherethe nucleic acid can contain combinations of deoxyribo- andribonucleotides, and combinations of bases including uracil, adenine,thymine, cytosine, guanine, inosine, xanthine hypoxanthine, isocytosineand isoguanine. Nucleic acids can be obtained by chemical synthesismethods or by recombinant methods. A particular nucleic acid sequencecan encompass conservatively modified variants thereof (e.g., codonsubstitutions), alleles, orthologs, single nucleotide polymorphisms(SNPs), and complementary sequences as well as the sequence explicitlyindicated.

“Polymerase Chain Reaction (PCR)” refers to the enzymatic reaction inwhich DNA fragments are synthesized and amplified from a substrate DNAin vitro. The reaction typically involves the use of two syntheticoligonucleotide primers, which are complementary to nucleotide sequencesin the substrate DNA which are separated by a short distance of a fewhundred to a few thousand base pairs, and the use of a thermostable DNApolymerase. The chain reaction consists of a series of 10 to 40 cycles.In each cycle, the substrate DNA is first denatured at high temperature.After cooling down, synthetic primers which are present in vast excess,hybridize to the substrate DNA to form double-stranded structures alongcomplementary nucleotide sequences. The primer-substrate DNA complexeswill then serve as initiation sites for a DNA synthesis reactioncatalyzed by a DNA polymerase, resulting in the synthesis of a new DNAstrand complementary to the substrate DNA strand. The synthesis processis repeated with each additional cycle, creating an amplified product ofthe substrate DNA.

“Primer,” as used herein, refers to an oligonucleotide capable of actingas a point of initiation for DNA synthesis under suitable conditions.Suitable conditions include those in which hybridization of theoligonucleotide to a template nucleic acid occurs, and synthesis oramplification of the target sequence occurs, in the presence of fourdifferent nucleoside triphosphates and an agent for extension (e.g., aDNA polymerase) in an appropriate buffer and at a suitable temperature.

“Probe” and “fluorescent generation probe” are synonymous and refer toeither a) a sequence-specific oligonucleotide having an attachedfluorophore and/or a quencher, and optionally a minor groove binder orb) a DNA binding reagent, such as, but not limited to, SYBR® Green dye.

“Quencher” refers to a molecule or part of a compound, which is capableof reducing the emission from a fluorescent donor when attached to or inproximity to the donor. Quenching may occur by any of several mechanismsincluding fluorescence resonance energy transfer, photo-induced electrontransfer, paramagnetic enhancement of intersystem crossing, Dexterexchange coupling, and exciton coupling such as the formation of darkcomplexes.

The term “RNase H PCR (rhPCR)” refers to a PCR reaction which utilizes“blocked” oligonucleotide primers and an RNase H enzyme. “Blocked”primers contain at least one chemical moiety (such as, but not limitedto, a ribonucleic acid residue) bound to the primer or otheroligonucleotide, such that hybridization of the blocked primer to thetemplate nucleic acid occurs, without amplification of the nucleic acidby the DNA polymerase. Once the blocked primer hybridizes to thetemplate or target nucleic acid, the chemical moiety is removed bycleavage by an RNase H enzyme, which is activated at a high temperature(e.g., 50° C. or greater). Following RNase H cleavage, amplification ofthe target DNA can occur.

The term “discrimination domain” can be the same or different as thecleavage domain. The discrimination domain is the position of thepotential mutation site, and the enzyme will only cleave at the cleavagesite if the criteria at the discrimination domain are met. For example,in one embodiment RNase H2 will cleave or not cleave a double-strandedtarget at the RNA residue (cleavage domain), depending on whether amutation exists at the discrimination domain.

In one embodiment, the 3′ end of a blocked primer can comprise themoiety rDDDDMx, wherein relative to the target nucleic acid sequence,“r” is an RNA residue, “D” is a complementary DNA residue, “M” is amismatched DNA residue, and “x” is a C3 spacer. A C3 spacer is a short3-carbon chain attached to the terminal 3′ hydroxyl group of theoligonucleotide, which further inhibits the DNA polymerase from bindingbefore cleavage of the RNA residue.

The methods described herein can be performed using any suitable RNase Henzyme that is derived or obtained from any organism. Typically, RNaseH-dependent PCR reactions are performed using an RNase H enzyme obtainedor derived from the hyperthermophilic archaeon Pyrococcus abyssi (P.a.),such as RNase H2. Thus, in one embodiment, the RNase H enzyme employedin the methods described herein desirably is obtained or derived fromPyrococcus abyssi, preferably an RNase H2 obtained or derived fromPyrococcus abyssi. In other embodiments, the RNase H enzyme employed inthe methods described herein can be obtained or derived from otherspecies, for example, Pyrococcus furiosis, Pyrococcus horikoshii,Thermococcus kodakarensis, or Thermococcus litoralis.

The following examples further illustrate the invention but should notbe construed as in any way limiting its scope.

Example 1

This example demonstrates an enhanced rhPCR assay that utilizes a highlydiscriminatory DNA polymerase and RNase H2 for discrimination

To demonstrate the utility of these new assay designs, rhPrimers andstandard allele-specific primers were designed against rs113488022, theV600E mutation in the human BRAF gene. These primers were tested in PCRand rhPCR with WT or H784Q mutant Taq polymerase. Primers utilized inthese assays were as shown in Table 1 (SEQ ID NOs: 1-7).

TABLE 1 Sequence of oligonucleotides employed in SNP discriminationassay described in Example 1. Name Sequence SEQ ID NO. Forward non-GCTGTGATTTTGGTCTAGCTACAG SEQ ID NO. 1 discriminating primer ForwardAllele GCTGTGATTTTGGTCTAGCTACAGT SEQ ID NO. 2 1 ASP1 ASPCR primerForward Allele GCTGTGATTTTGGTCTAGCTACAGA SEQ ID NO. 3 2 ASP2 ASPCRprimer Probe FAM-TCCCATCAG-ZEN- SEQ ID NO. 4 TTTGAACAGTTGTCTGGA-IBFQrs113488022 GCTGTGATTTTGGTCTAGCTACAGTgAA SEQ ID NO. 5 Allele 1 ATG-xForward ASP1 rhPrimer rs113488022 GCTGTGATTTTGGTCTAGCTACAGAgAA SEQ IDNO. 6 Allele 2 ATG-x Forward ASP2 rhPrimer ReverseGCCCTCAATTCTTACCATCCACAAAaTG SEQ ID NO. 7 rhPrimer GAA-x Nucleic acidsequences are shown 5′-3′. DNA is uppercase, RNA is lowercase. Locationof potential mismatch is underlined. ZEN = internal ZEN ™ quencher (IDT,Coralville, IA), FAM = 6-carboxyfluorescein, IBFQ = Iowa Black ® FQ(fluorescence quencher, IDT, Coralville, IA), and x = C3 propanediolspacer block

10 μL reaction volumes were used in these assays. To perform thereaction, 5 μL of 2× Integrated DNA Technologies (IDT) (Coralville,Iowa) rhPCR genotyping master mix (containing dNTPs, H784Q mutant or WTTaq DNA polymerase, stabilizers, and MgCl₂) was combined with 200 nM (2pmol) of either of the allelic primers. 200 nM (2 pmol) of the probe, aswell as 200 nM (5 pmol) of the reverse primer were also added.Additionally, 2.5 mU (5.25 fmol/0.53 nM) of P.a. RNase H2 and 1000copies of synthetic gBlock™ (Integrated DNA Technologies, Coralville,Iowa) template (1000 copies Allele 1, 500 copies allele 1+500 copiesallele 2 (heterozygote), or 1000 copies Allele 2 (for gBlock™ sequences,see Table 2, SEQ ID NOs: 8-9) were added to the reaction mix. Thereaction was thermocycled on a Bio-Rad™ CFX384™ Real-time system.Cycling conditions were as follows: 953:00−(950:10−650:30)×65 cycles.Each reaction was performed in triplicate.

TABLE 2 Synthetic gBlock templates for Example 1 assay SEQ Name SequenceID NO. rs113488022 AAAAAATAAGAACACTGATTTTTGTGAAT SEQ ID gBlockACTGGGAACTATGAAAATACTATAGTTGA NO. 8 Template 1GACCTTCAATGACTTTCTAGTAACTCAGCA GCATCTCAGGGCCAAAAATTTAATCAGTGGAAAAATAGCCTCAATTCTTACCATCCAC AAAATGGATCCAGACAACTGTTCAAACTGATGGGACCCACTCCATCGAGATTTC A CTGT AGCTAGACCAAAATCACCTATTTTTACTGTGAGGTCTTCATGAAGAAATATATCTGAGG TGTAGTAAGTAAAGGAAAACAGTAGATCTCATTTTCCTATCAGAGCAAGCATTATGAAG AGTTTAGGTAAGAGATCTAATTTCTATAATTCTGTAATATAATATTCTTTAAAACATAGT ACTTCATCTTTCCTCTTA rs113488022AAAAAATAAGAACACTGATTTTTGTGAAT SEQ ID gBlockACTGGGAACTATGAAAATACTATAGTTGA NO. 9 Template 2GACCTTCAATGACTTTCTAGTAACTCAGCA GCATCTCAGGGCCAAAAATTTAATCAGTGGAAAAATAGCCTCAATTCTTACCATCCAC AAAATGGATCCAGACAACTGTTCAAACTGATGGGACCCACTCCATCGAGATTTC T CTGT AGCTAGACCAAAATCACCTATTTTTACTGTGAGGTCTTCATGAAGAAATATATCTGAGG TGTAGTAAGTAAAGGAAAACAGTAGATCTCATTTTCCTATCAGAGCAAGCATTATGAAG AGTTTAGGTAAGAGATCTAATTTCTATAATTCTGTAATATAATATTCTTTAAAACATAGT ACTTCATCTTTCCTCTTA Nucleic acid sequencesare shown 5′-3′. Location of SNPs are shown bold and underlined.

Cq Results of the experiment are shown in Table 3. This data shows thatthe mismatch discrimination of the assay system increases with rhPCRover ASPCR with WT Taq polymerase, and that the discrimination isenhanced by the use of the H784Q Taq polymerase.

TABLE 3 Resulting Cq values WT Taq H784Q Allele Allele Allele Allele 1Het 2 NTC 1 Het 2 NTC Non discrmin 29.3 29.3 29.4 >65 30.6 30.6 30.8 >65ASP1 ASPCR 30.2 30.2 31.4 >65 29.2 32.5 40.3 >65 ASP2 ASPCR 36.7 30.529.4 >65 44.2 31.7 30.8 >65 ASP1 rhPCR 30.9 32.1 38.2 >65 31.9 31.449.2 >65 ASP2 rhPCR 39.3 31.0 30.8 >65 43.4 33.9 32.5 >65 All numbers inthis table represent Cq values obtained from the CFX384 ™ instrument(Bio-Rad ™, Hercules, CA).

Example 2

The following example demonstrates an enhanced rhPCR assay that utilizesa highly discriminatory DNA polymerase and RNase H2 for discrimination.

In order to demonstrate that this new assay design could function,rhPrimers and standard allele-specific primers were designed againstrs113488022, the V600E mutation in the human BRAF gene. These primerswere tested in PCR and rhPCR with H784Q mutant Taq polymerase. Primersutilized in these assays were as shown in Table 4 (SEQ ID NOs: 1, 4 and10-12).

TABLE 4 Sequence of oligonucleotides employed in SNP discriminationassay described in Example 2 SEQ Name Sequence ID NO. Forward non-GCTGTGATTTTGGTCTAGCTACAG SEQ ID discrimin primer NO. 1 ProbeFAM-TCCCATCAG-ZEN- SEQ ID TTTGAACAGTTGTCTGGA-IBFQ NO. 4 rs113488022GCTGTGATTTTGGTCTAGCTACAGTg SEQ ID Allele 1 Forward AxxTG NO. 10 dxxdrhPrimer rs113488022 GCTGTGATTTTGGTCTAGCTACAGAg SEQ ID Allele 2 ForwardAxxTG NO. 11 dxxd rhPrimer Reverse rhPrimer GCCCTCAATTCTTACCATCCACAAAaSEQ ID TGGAA-x NO. 12 Nucleic acid sequences are shown 5′-3′. DNA isuppercase, RNA is lowercase. Location of potential mismatch isunderlined. ZEN = internal Zen ® fluorescent quencher (IDT, Coralville,IA). FAM = 6-carboxyfluorescein, IBFQ = Iowa Black FQ (fluorescencequencher), and x = C3 propanediol spacer.

10 μL reaction volumes were used in these assays. To perform thereaction, 5 μL of 2× Integrated DNA Technologies (IDT) (Coralville,Iowa) rhPCR genotyping master mix (containing dNTPs, H784Q mutant DNApolymerase, stabilizers, and MgCl₂) was combined with 200 nM (2 pmol) ofeither of the allelic primers. 200 nM (2 pmol) of the probe, as well as200 nM (5 pmol) of the reverse primer were also added. Additionally, 7.5mU (15.75 fmol/1.58 nM), 50 mU (105 fmol/10.5 nM) or 200 mU (420 fmol/42nM) of P.a. RNase H2 and 5e4 copies of synthetic gBlock™ (Integrated DNATechnologies, Coralville, Iowa) template (1e5 copies Allele 1, 5e4copies allele 1+5e4 copies allele 2 (heterozygote), or 1e5 copies Allele2 (for gBlock™ sequences, see Table 2, SEQ ID NOs: 8-9) were added tothe reaction mix. The reaction was thermocycled on a Bio-Rad™ CFX384™Real-time system. Cycling conditions were as follows:95^(3:00)−(95^(0:10)−65^(0:30))×65 cycles. Each reaction was performedin triplicate.

Cq Results of the experiment are shown in Table 5. This data shows thatthe mismatch discrimination of the assay system increases with rhPCRover ASPCR with WT Taq polymerase, and that the discrimination isenhanced by the use of the H784Q Taq polymerase.

TABLE 5 Resulting Cq values Averages Allele 1 Het Allele 2 NTC ΔCqUnblocked  7.5 mU 21.9 22.3 22.1 >75  50 mU 22.7 22.5 22.7 >75 200 mU21.8 21.8 21.9 >75 AgAxxTG  7.5 mU 43.7 25.6 24.6 >75 19.1  50 mU 50.324.5 23.5 >75 26.8 200 mU 48.5 25.2 24.1 >75 24.4 TgAxxTG  7.5 mU 25.126.3 42.5 >75 17.4  50 mU 24.2 25.4 41.0 >75 16.9 200 mU 22.9 23.837.2 >75 14.3 All numbers in this table represent Cq values obtainedfrom the CFX384 ™ instrument (Bio-Rad ™, Hercules, CA).

The delta Cq values were significantly higher than the ones obtainedwith the Gen 1 versions of these primers, indicating that there is anadvantage to this primer design, as seen before in rhPCR.

Example 3

The following example illustrates the heightened reliability ofuniversal assays using a DNA polymerase with a high mismatchdiscrimination.

To demonstrate that the disclosed assays can function in a universalformat and that they are significantly improved with a polymerase withhigh mismatch discrimination, “universal” assay primers were designedagainst rs351855, the G338R mutation in the human FGFR4 gene. This“universal” assay design has numerous advantages, including the abilityto multiplex the allele-specific rhPrimers and obtain multiple-colorreadouts. Primers utilized in this assay were as shown in Table 6 (SEQID NOs: 13-18).

TABLE 6 Sequences of oligonucleotides employed in “universal” SNPdiscrimination assay SEQ Name Sequence ID NO. UniversalCGCCGCGTATAGTCCCGCGTAAA SEQ ID Forward NO. 13 primer Probe 1FAM-C+CATC+A+C+CGTG+CT-IBFQ SEQ ID (FAM) NO. 14 Probe 2HEX-CAATC+C+C+CGAG+CT-IBFQ SEQ ID (HEX) NO. 15 rs351855GCCCATGTCCCAGCGAACCATCACCGTGCTA SEQ ID Allele 1 GCCCTCGATACAGCCCgGCCAC-xNO. 16 Forward primer rs351855 GCCCATGTCCCAGCGAACAATCCCCGAGCTG SEQ IDAllele 2 CCCTCGATACAGCCTgGCCAC-x NO. 17 Forward primer ReverseGCGGCCAGGTATACGGACATcATCCA-x SEQ ID primer NO. 18 Nucleic acid sequencesare shown 5′-3′. DNA is uppercase, RNA is lowercase. Location ofpotential mismatch is underlined. LNA residues are designated with a +.FAM = 6-carboxyfluorescein, HEX= 6-carboxy-2′,4,4′,5′,7,7′-hexachlorofluorescein, IBFQ = Iowa Black FQ(fluorescence quencher), and x = C3 propanediol spacer block.

10 μL reaction volumes were used in these assays. To perform thereaction, 5 μL of 2× Integrated DNA Technologies (IDT) (Coralville,Iowa) rhPCR genotyping master mix (containing dNTPs, mutant or WT TaqDNA polymerase, stabilizers, and MgCl₂) was combined with 50 nM (500fmol) of each of the two allelic primers. 250 nM (2.5 pmol) of each ofthe two probes, as well as 500 nM (5 pmol) of the Universal Forwardprimer and 500 nM (5 pmol) of the reverse primer were also added.Additionally, 2.5 mU (5.25 fmol/0.53 nM) of P.a. RNase H2 and 1000copies of synthetic gBlock™ (Integrated DNA Technologies, Coralville,Iowa) template (1000 copies Allele 1, 500 copies allele 1+500 copiesallele 2 (heterozygote), or 1000 copies Allele 2 (for gBlock™ sequences,see Table 7, SEQ ID NOs: 19-20) were added to the reaction mix. Thereaction was thermocycled on a Bio-Rad™ CFX384™ Real-time system.Cycling conditions were as follows: 95^(3:00)−(95^(0:10)−60^(:30))×3cycles−(95^(0:10)−65^(0:30))×65 cycles. Each reaction was performed intriplicate. Fluorescence reads were taken after a total of 50 cycleswere completed. Fluorescence values were plotted on the FAM and HEXaxis, and results are shown in FIGS. 2a and 2b .

TABLE 7 Synthetic gBlock templates for Example 3 SEQ ID Name SequenceNO. rs351855 GTTGGGAGCTGGGAGGGACTGAGTTAGGG SEQ ID gBlockTGCACGGGGCGGCCAGTCTCACCACTGAC NO. 19 Template 1CAGTTTGTCTGTCTGTGTGTGTCCATGTGC GAGGGCAGAGGAGGACCCCACATGGACCGCAGCAGCGCCCGAGGCCAGGTATACGGA CATCATCCTGTACGCGTCGGGCTCCCTGGCCTTGGCTGTGCTCCTGCTGCTGGCC G GGCT GTATCGAGGGCAGGCGCTCCACGGCCGGCACCCCCGCCCGCCCGCCACTGTGCAGAAG CTCTCCCGCTTCCCTCTGGCCCGACAGGTACTGGGCGCATCCCCCACCTCACATGTGAC AGCCTGACTCCAGCAGGCAGAACCAAGTCTCCCACTTTGCAGTTCTCCCTGGAGTCAGG CTCTTCCGGCAAGTCAAGCT rs351855GTTGGGAGCTGGGAGGGACTGAGTTAGGG SEQ ID gBlockTGCACGGGGCGGCCAGTCTCACCACTGAC NO. 20 Template 2CAGTTTGTCTGTCTGTGTGTGTCCATGTGC GAGGGCAGAGGAGGACCCCACATGGACCGCAGCAGCGCCCGAGGCCAGGTATACGGA CATCATCCTGTACGCGTCGGGCTCCCTGGCCTTGGCTGTGCTCCTGCTGCTGGCC A GGCT GTATCGAGGGCAGGCGCTCCACGGCCGGCACCCCCGCCCGCCCGCCACTGTGCAGAAG CTCTCCCGCTTCCCTCTGGCCCGACAGGTACTGGGCGCATCCCCCACCTCACATGTGAC AGCCTGACTCCAGCAGGCAGAACCAAGTCTCCCACTTTGCAGTTCTCCCTGGAGTCAGG CTCTTCCGGCAAGTCAAGCT Nucleic acidsequences are shown 5′-3′. Location of SNPs are shown bold andunderlined.

The results illustrate that the mismatch discrimination betweenhomozygotes is sufficient with both polymerases, although the resultingdata using the WT Taq demonstrate that it is more difficult to make anallelic call. Importantly, however, the WT Taq polymerase cannotefficiently discriminate heterozygotes from homozygotes, and places themtoo close to the allele 1 and 2 signals (FIG. 2a ). In contrast, thesignal from the heterozygotes in the assays utilizing the mutant Taqpolymerase are easily distinguishable from the homozygotes (FIG. 2b ).

The importance of the mutant Taq can be further understood whenexamining the Cq values from this example (Table 8). The data show thatnot only does the H784Q Taq mutant increase mismatch discriminationdramatically, but the Cqs of the NTCs decrease from the low-to-mid 50s,to greater than the number tested in the assay (>65). From thisexperiment, it is shown that allele identity can be determined from Cqvalues as well as end-point fluorescence.

TABLE 8 Cq and delta Cq data for the experiment described in Example 3WT Taq H784Q Template FAM HEX Delta Cq FAM HEX Delta Cq Allele 1 32.931.3 −1.6 37.5 56.8 19.3 Allele 1 31.9 31.1 −0.8 36.2 51.4 15.2 Allele 131.8 31.0 −0.9 36.6 54.3 17.8 Heterozygote 33.0 29.4 −3.5 38.7 37.8 −0.9Heterozygote 32.8 29.7 −3.1 38.7 38.2 −0.5 Heterozygote 33.2 30.0 −3.339.9 39.1 −0.8 Allele 2 35.1 29.1 6.0 50.6 36.5 14.1 Allele 2 34.7 29.35.4 52.1 36.6 15.5 Allele 2 34.5 29.0 5.5 50.7 36.1 14.6 NTC 51.8 56.1— >65 >65 — NTC 52.8 56.1 — >65 >65 — NTC 52.1 50.7 — >65 >65 — Allnumbers in this table represent Cq and delta Cq values values obtainedfrom the CFX384 instrument (Bio-Rad ™, Hercules, CA).

Example 4

The following example illustrates the detection of rare allelic variantswith the assay designs of the present invention. To demonstrate theutility of these new assay designs to detect rare allelic variants,previously described second generation rhPrimers (rdxxdm) were utilizedagainst rs113488022, the V600E mutation in the human BRAF gene (seeTable 4; SEQ ID NOs: 1, 4 and 10-12).

10 μL reaction volumes were used in these assays. To perform thereaction, 5 μL of 2× Integrated DNA Technologies (IDT) (Coralville,Iowa) rhPCR genotyping master mix (containing dNTPs, H784Q mutant or WTTaq DNA polymerase, stabilizers, and MgCl₂) was combined with 200 nM (2pmol) of either of the allelic primers, or the non-discriminatoryforward primer. 200 nM (2 pmol) of the probe, as well as 200 nM (5 pmol)of the reverse primer were also added. Additionally, 50 mU (105fmol/10.5 nM) of P.a. RNase H2 and 50,000 copies of synthetic gBlock™(Integrated DNA Technologies, Coralville, Iowa) match template, wascombined with either 0, 50, or 500 copies of the opposite allele (forgBlock™ sequences, see Table 6, SEQ ID NOs: 16-17) were added to thereaction mix. The reaction was thermocycled on a Bio-Rad™ CFX384™Real-time system. Cycling conditions were as follows:95^(3:00)−(95^(0:10)−60^(0:30))×65 cycles. Each reaction was performedin triplicate.

Data for the WT polymerase is shown in Table 9, and for the H784Q mutantTaq polymerase in Table 10. One of the advantages of this system forrare allele detection over “conventional” rhPCR is the ability toutilize a single amount of RNase H2 for both alleles. This advantagehalves the potential requirement for determining the enzyme amountrequired for cleavage.

TABLE 9 Average Cq and delta Cq values for the rare allele experimentwith the WT Taq polymerase described in Example 4. Back-ground 50,00050,000 50,000 0 0 0 Target 500 50 0 500 50 0 SEQ Non-discrimin 22.9 23.122.8 30.4 34.2 >65 ID No. 1 SEQ . . . TgAxxTG 31.6 34.5 36.1 31.636.0 >65 ID NO. 10 SEQ . . . AgAxxTG 29.1 29.1 29.9 30.8 34.4 >65 ID NO.11 All numbers in this table represent Cq and delta Cq values valuesobtained from the CFX384 instrument (Bio-Rad ™, Hercules, CA). DNA isuppercase, RNA is lowercase. Location of potential mismatch isunderlined. x = internal C3 propanediol spacer block.

TABLE 10 Average Cq and delta Cq values for the rare allele experimentwith the H784Q mutant Taq polymerase described in Example 4. Back-ground50,000 50,000 50,000 0 0 0 Target 500 50 0 500 50 0 SEQ Non-discrimin22.7 23.2 23.2 31.5 34.2 >65 ID No. 1 SEQ . . . TgAxxTG 33.8 36.6 47.333.1 36.4 >65 ID NO. 10 SEQ . . . AgAxxTG 32.1 35.2 38.8 32.0 35.5 >65ID NO. 11 All numbers in this table represent Cq and delta Cq valuesobtained from the CFX384 instrument (Bio-Rad ™, Hercules, CA). DNA isuppercase, RNA is lowercase. Location of potential mismatch isunderlined. x = internal C3 propanediol spacer block.

The data clearly shows that the H784Q DNA polymerase allows fordetection of 50 copies of target in a 50,000 copies of background DNA (a1:1000 discrimination level) for the mutant A allele of rs113488022,with a delta Cq of over 11 cycles. While only slightly more than 3cycles was observed for the T allele in this assay, this was asignificant improvement over the WT Taq polymerase, which did not showany discrimination for the T allele, and only a delta of 3 cycles forthe A allele.

Example 5

This example demonstrates successful allelic discrimination with the useof a universal rhPCR genotyping assay and Integrated DNA Technologies(IDT) (Coralville, Iowa) rhPCR genotyping master mix, and the robuststability of the reaction components. To demonstrate the robust natureof the assay design and mixture components, universal primers weredesigned against rs4657751, a SNP located on the human Chromosome 1 (SeeTable 11, SEQ ID NOs: 14, 21-25).

Identical universal rhPCR genotyping reactions were set up in two whiteHard-Shell® 384-well skirted PCR plates (Bio-Rad, Hercules, Calif.) onthe Bio-Rad CFX384 Touch™ Real-Time PCR Detection System with 10 μLfinal volume. Each well contained the rhPCR assay primers (150 nM ofrs4657751 Allele Specific Primer 1 (SEQ ID NO: 23), 150 nM of rs4657751Allele Specific Primer 2 (SEQ ID NO: 24), and 500 nM rs4657751 LocusSpecific Primer (SEQ ID NO: 25). Reactions contained universal reporteroligos at the following concentrations: 250 nM of universal FAM probe(SEQ ID NO: 14), 450 nM of universal Yakima Yellow® (SEQ ID NO: 22)probe, and 1000 nM of universal forward primer (SEQ ID NO: 21), and 5 μLof 2× Integrated DNA Technologies (IDT) (Coralville, Iowa) rhPCRgenotyping master mix (containing dNTPs, a mutant H784Q Taq polymerase(see Behlke, et al. U.S. 2015/0191707), chemically modified Pyrococcusabyssi RNase H2 (See Walder et al. UA20130288245A1), stabilizers, andMgCl₂).

gBlocks® Gene Fragments (Integrated DNA Technologies, Inc., Coralville,Iowa) containing either allele of the rs4657751 SNP were utilized as thesource of template DNA (See Table 12, SEQ ID NOs: 26 and 27). Each wellcontained template representing one of three possible genotypes: allele1 homozygote (1000 copies rs4657751 Allele 1 gBlock® template (SEQ IDNO: 26)), allele 2 homozygote (1000 copies rs4657751 Allele 2 gBlock®template (SEQ ID NO: 27)), or heterozygote (mix of 500 copies ofrs4657751 Allele 1 gBlock® template (SEQ ID NO: 26) and 500 copies ofrs4657751 Allele 2 gBlock® template (SEQ ID NO: 27)). Template or waterfor the no template control (NTC) reactions were added into threereplicate wells of two individual plates. The reactions underwent thefollowing cycling protocol: 95° C. for 10 minutes, then 45 cycles of 95°C. for 10 seconds and 60° C. for 45 seconds.

TABLE 11 Sequences of oligonucleotides used in Example 5 SEQ ID NameSequence NO. Universal CGGCCCATGTCCCAGCGAA SEQ ID Forward NO. 21 primerProbe 1 FAM-C+CATC+A+C+CGTG+CT-IBFQ SEQ ID (FAM) NO. 14 Probe 2Yak-CAATC+C+C+CGAG+CT-IBFQ SEQ ID (Yakima NO. 22 Yellow) rs4657751GCCCATGTCCCAGCGAACCATCACCGTGC SEQ ID Allele 1TACTTCCCACACCCTCATATCuTGTTA-x NO. 23 Forward primer rs4657751GCCCATGTCCCAGCGAACAATCCCCGAGC SEQ ID Allele 2TCTTACTTCCCACACCCTCATATAuTGTTA-x NO. 24 Forward primer rs4657751GCGCTAAGTAAACATTCCTGATTGCAaCTT SEQ ID Reverse AT-x NO. 25 primer Nucleicacid sequences are shown 5′-3′. DNA is uppercase, RNA is lowercase.Location of potential mismatch is underlined. LNA residues aredesignated with a +. FAM = 6-carboxyfluorescein, Yak = Yakima Yellow(3-(5,6,4′,7′-tetrachloro-5′-methyl-3′,6′-dipivaloylfluorescein-2-yl)),IBFQ = Iowa Black FQ (fluorescence quencher), and x = C3 propanediolspacer block.

TABLE 12 Synthetic gBlock ® templates used in Example 5. SEQ NameSequence ID NO. rs4657751 GATTTTTTTTTTTTGGCATTTCTTCTTAGAT SEQ Allele 1TTCTATCTCCTAACATAGGATCACTTATTT ID NO. 26 gBlockGTGAAATTATTTGTATACCTTTTTTATGGA template GTGATGATGTGATACAAATTCTATCCTTAAGGATATAAGAACATCTTTTCTTTATATTAG GATTTTTCTGGACCCATGAGTTACATGCTTACTTCCCACACCCTCATATCTTGTTTAAAT TTGTAGAATTAAATTCATAGGTAATTATTTCTGAAACTTCTTCCCTGTGTGAGCAATCTA AATAATTATTACAATGCCTTAAGTTGCAATCAGGAATGTTTACTTAGCACAGACTTTTTT CCCCACTACTGCACTCAAAGGATAACAGATATATGGCAAATCTAACCATATTCTTTGTC CTTTGTCCATGTTGCGGAGGGAAGCTCATCAGTGGGGCCACGAGCTGAGTGCGTCCTGT CACTCCACTCCCATGTCCCTTGGGAAGGTC TGAGACTAGGGrs4657751 GATTTTTTTTTTTTGGCATTTCTTCTTAGAT SEQ Allele 2TTCTATCTCCTAACATAGGATCACTTATTT ID NO. 27 gBlockGTGAAATTATTTGTATACCTTTTTTATGGA template GTGATGATGTGATACAAATTCTATCCTTAAGGATATAAGAACATCTTTTCTTTATATTAG GATTTTTCTGGACCCATGAGTTACATGCTTACTTCCCACACCCTCATATATTGTTTAAAT TTGTAGAATTAAATTCATAGGTAATTATTTCTGAAACTTCTTCCCTGTGTGAGCAATCTA AATAATTATTACAATGCCTTAAGTTGCAATCAGGAATGTTTACTTAGCACAGACTTTTTT CCCCACTACTGCACTCAAAGGATAACAGATATATGGCAAATCTAACCATATTCTTTGTC CTTTGTCCATGTTGCGGAGGGAAGCTCATCAGTGGGGCCACGAGCTGAGTGCGTCCTGT CACTCCACTCCCATGTCCCTTGGGAAGGTC TGAGACTAGGGNucleic acid sequences are shown 5′-3′. DNA is uppercase. The locationof the SNP is underlined.

One reaction plate was cycled immediately (0 hr benchtop hold) and onereaction plate was held at room temperature for 2 days (48 hr benchtophold) to demonstrate reaction stability over time. Allelicdiscrimination analysis was performed using Bio-Rad CFX Manager 3.1software (Bio-Rad, Hercules, Calif.). FAM and Yakima Yellow fluorophoreswere detected in each well. For both fluorophores the baseline cycleswere set to begin at cycle 10 and end at cycle 25. Fluorescence signal(RFU) in each well at the end of 45 cycles was used to generate anallelic discrimination plot and genotypes were determined with auto-callfeatures of the analysis software. Identical performance was obtainedwith the immediate run (FIG. 3A) and 48 hour hold plate (FIG. 3B),demonstrating robust stability of the reaction components. Each sampleis assigned the correct genotyping call and samples of the same genotypeare tightly clustered together. The heterozygote cluster is separatedfrom both of the homozygous clusters by an approximate 45 degree angle,indicating excellent allelic specificity of the universal rhPCRgenotyping assays and master mix.

Example 6

The following example compares the performance of the genotyping methodsof the present invention versus traditional 5′ nuclease genotyping assaymethods (Taqman™).

The rs1799865 SNP in the CCR2 gene was selected, and rhPCR genotypingprimers as well as an rs1799865 5′ nuclease assay (Thermo-Fisher(Waltham, Mass.)), were designed and obtained. Sequences for thers1799865 rhPCR genomic SNP assay are shown in Table 14 (SEQ ID NOs: 14,21, 22, and 28-30). Thermo-Fisher 5′ nuclease primer/probe (Taqman™)sequences are not published, and therefore are not included in thisdocument.

Reactions were performed in 10 μL volumes, containing 10 ng Coriellgenomic DNA (Camden, N.J.), 250 nM of universal FAM probe (SEQ ID NO:14), 450 nM of universal Yakima Yellow® (SEQ ID NO: 22) probe, 1000 nMof universal forward primer (SEQ ID NO: 21), 150 nM of the twoallele-specific forward primers (SEQ ID NOs: 28 and 29), 500 nM of thereverse primer (SEQ ID NO: 30), and 5 μL of 2× Integrated DNATechnologies (IDT) (Coralville, Iowa) rhPCR genotyping master mix(containing dNTPs, a mutant H784Q Taq polymerase (see Behlke, et al.U.S. 2015/0191707), chemically modified Pyrococcus abyssi RNase H2 (SeeWalder et al. UA20130288245A1), stabilizers, and MgCl₂).

PCR was performed on Life Technologies (Carlsbad, Calif.) QuantStudio™ 7Flex real-time PCR instrument using the following cycling conditions: 10mins at 95° C. followed by 50 cycles of 95° C. for 10 seconds and 60° C.for 45 seconds. End-point analysis of each of the plates was performedafter 45 cycles with the QuantStudio™ Real-Time PCR Software v1.3(Carlsbad, Calif.).

TABLE 14 Sequences of oligonucleotides used for the rs1799865 genotypingassay in Example 6. SEQ Name Sequence ID NO. UniversalCGGCCCATGTCCCAGCGAA SEQ Forward ID NO. 21 primer Probe 1FAM-C+CATC+A+C+CGTG+CT-IBFQ SEQ (FAM) ID NO. 14 Probe 2Yak-CAATC+C+C+CGAG+CT-IBFQ SEQ (Yakima ID NO. 22 Yellow) rs1799865GCCCATGTCCCAGCGAACCATCACCGTG SEQ Allele 1 CTTTCTCTTCTGGACTCCCTATAATaTTGTID NO. 28 Forward G-x primer rs1799865 GCCCATGTCCCAGCGAACAATCCCCGAG SEQAllele 2 CTTTCTCTTCTGGACTCCCTATAACaTTGT ID NO. 29 Forward G-x primerrs1799865 GCGGATTGATGCAGCAGTGAgTCATG-x SEQ Reverse ID NO. 30 primerNucleic acid sequences are shown 5′-3′. DNA is uppercase, RNA islowercase. LNA residues are designated with a +. Location of potentialmismatch is underlined. FAM = 6-carboxyfluorescein, Yak = Yakima Yellow(3-(5,6,4′,7′-tetrachloro-5′-methyl-3′,6′-dipivaloylfluorescein-2-yl)),IBFQ = Iowa Black FQ (fluorescence quencher), and x = C3 propanediolspacer block.

FIGS. 4A and 4B show a side-by-side comparison of the resulting allelicdiscrimination plots. The rhPCR Genotyping Assay (FIG. 4B) achievedhigher fluorescence signal compared to a traditional 5′-nucleasegenotyping assay (FIG. 4A) while showing concordant results. The highersignal and minimal non-specific amplification from NTC in the rhPCRassay allow better cluster separation and accurate genotype calls.

Example 7

The following example illustrates the present methods allowing fordetection and analysis of tri-allelic SNP. The rs72558195 SNP is presentin the CYP2C8 gene, and has three potential genotypes. This SNP wasselected for analysis with the rhPCR genotyping system.

Conventional workflow of interrogating tri-allelic SNP, as illustratedin FIGS. 5A and 5B, involves running a pair of assays using the samesamples, manual calling, and comparing the paired assay result to obtainthe true genotype of samples.

To demonstrate that such a system can function with the universal rhPCRgenotyping system, reactions were set up in a white Hard-Shell® 384-wellskirted PCR plates (Bio-Rad, Hercules, Calif.) on the Life Technologies(Carlsbad, Calif.) QuantStudio™ 7 Flex real-time PCR with 10 μL finalvolume. Each well contained the rhPCR assay primers (See Table 16, SEQID NOs: 14, 21, 22, 31-33). Specifically, 150 nM of rs72558195 G:AAllele Specific Primer 1 (SEQ ID NO: 31) and 150 nM of rs72558195 G:AAllele Specific Primer 2 (SEQ ID NO: 32), or 150 nM of rs72558195 G:AAllele Specific Primer 1 (SEQ ID NO: 31) and 150 nM of rs72558195 G:CAllele Specific Primer 3 (SEQ ID NO: 33) as well as 500 nM rs72558195Locus Specific Primer (SEQ ID NO: 34) were included in the reactions.

Reactions contained universal reporter oligos at the followingconcentrations: 250 nM of universal FAM probe (SEQ ID NO: 14), 450 nM ofuniversal Yakima Yellow® (SEQ ID NO: 22) probe, and 1000 nM of universalforward primer (SEQ ID NO: 21), 50 nM ROX internal standard, and 5 μL of2× Integrated DNA Technologies (IDT) (Coralville, Iowa) rhPCR genotypingmaster mix (containing dNTPs, a mutant H784Q Taq polymerase (see Behlke,et al. U.S. 2015/0191707), chemically modified Pyrococcus abyssi RNaseH2 (See Walder et al. UA20130288245A1), stabilizers, and MgCl₂).

TABLE 16 Sequences of oligonucleotides used for the rs72558195genotyping assay in Example 7. SEQ Name Sequence ID NO. UniversalCGGCCCATGTCCCAGCGAA SEQ ID Forward NO. 21 primer Probe 1FAM-C+CATC+A+C+CGTG+CT-IBFQ SEQ ID (FAM) NO. 14 Probe 2Yak-CAATC+C+C+CGAG+CT-IBFQ SEQ ID (Yakima NO. 22 Yellow) rs72558195:GCCCATGTCCCAGCGAACCATCACCGTGCTC SEQ ID G:A AlleleTCCGTTGTTTTCCAGAAACgATTTC-x NO. 31 1 Forward primer rs72558195:GCCCATGTCCCAGCGAACAATCCCCGAGCTC SEQ ID G:A AlleleTCCGTTGTTTTCCAGAAATgATTTC-x NO. 32 2 Forward primer rs72558195:GCCCATGTCCCAGCGAACAATCCCCGAGCTC SEQ ID G:C AlleleTCCGTTGTTTTCCAGAAAGgATTTC-x NO. 33 3 Forward Primer rs1135840GCAACCAAGTCTTCCCTACAACcTTGAT-x SEQ ID Reverse NO. 34 primer Nucleic acidsequences are shown 5′-3′. DNA is uppercase, RNA is lowercase. LNAresidues are designated with a +. Location of potential mismatch isunderlined. FAM = 6-carboxyfluorescein, Yak = Yakima Yellow(3-(5,6,4′,7′-tetrachloro-5′-methyl-3′,6′-dipivaloylfluorescein-2-yl)),IBFQ = Iowa Black FQ (fluorescence quencher), and x = C3 propanediolspacer block.

gBlocks® Gene Fragments (Integrated DNA Technologies, Inc., Coralville,Iowa) containing alleles of the rs72558195 SNP were utilized as thesource of template DNA (See Table 17, SEQ ID NOs: 35, 36 and 37). Eachwell contained template representing one of six possible genotypes:allele 1 homozygote (1000 copies rs72558195 Allele 1 gBlock® template(SEQ ID NO: 35)), allele 2 homozygote (1000 copies rs72558195 Allele 2gBlock® template (SEQ ID NO: 36)), allele 3 homozygote (1000 copiesrs72558195 Allele 2 gBlock® template (SEQ ID NO: 37)), heterozygote (mixof 500 copies of rs72558195 Allele 1 gBlock® template (SEQ ID NO: 35)and 500 copies of rs72558195 Allele 2 gBlock® template (SEQ ID NO: 36).heterozygote (mix of 500 copies of rs72558195 Allele 1 gBlock® template(SEQ ID NO: 35) and 500 copies of rs72558195 Allele 3 gBlock® template(SEQ ID NO: 37)). Template or water for the no template control (NTC)reactions were added into three replicate wells of two individualplates. The reactions underwent the following cycling protocol: 95° C.for 10 minutes, then 45 cycles of 95° C. for 10 seconds and 60° C. for45 seconds.

TABLE 17 gBlock ® sequences used in Example 7 SEQ Name Sequence ID NO.rs72558195 ACATCATTTTTATTGTATAAAAGCATTTTA SEQ ID Allele 1GTATCAATTTTCTCATTTTTAAACCAAGTC NO. 35 gBlockTTCCCTACAACCTTGAATAAATGGTTTCCA template AGGAAAATAAAATCTTGGCCTTACCTGGATCCATGGGGAGTTCAGAATCCTGAAGTTT TCATTGAATCTTTTCATCAGGGTGAGAAAATTCTGATCTTTATAATCAAATCGTTTCTG GAAAACAACGGAGCAGATCACATTGCAGGGAGCACAGCCCAGGATGAAAGTGGGAT CACAGGGTGAAGCTAAAGATTTAAAAATTTTTAAAAAAATTATTAAAAAATAAATATT TAAAAGATTTGCATTTGTTAAGACATAAAGGAAATTTAGAAATTTTAAACAATATCTT ACAAATTCCCCATGTGTCCAAA rs72558195ACATCATTTTTATTGTATAAAAGCATTTTA SEQ ID Allele 2GTATCAATTTTCTCATTTTTAAACCAAGTC NO. 36 gBlockTTCCCTACAACCTTGAATAAATGGTTTCCA template AGGAAAATAAAATCTTGGCCTTACCTGGATCCATGGGGAGTTCAGAATCCTGAAGTTT TCATTGAATCTTTTCATCAGGGTGAGAAAATTCTGATCTTTATAATCAAATCATTTCTG GAAAACAACGGAGCAGATCACATTGCAGGGAGCACAGCCCAGGATGAAAGTGGGAT CACAGGGTGAAGCTAAAGATTTAAAAATTTTTAAAAAAATTATTAAAAAATAAATATT TAAAAGATTTGCATTTGTTAAGACATAAAGGAAATTTAGAAATTTTAAACAATATCTT ACAAATTCCCCATGTGTCCAAA rs72558195ACATCATTTTTATTGTATAAAAGCATTTTA SEQ ID Allele 3GTATCAATTTTCTCATTTTTAAACCAAGTC NO. 37 gBlockTTCCCTACAACCTTGAATAAATGGTTTCCA template AGGAAAATAAAATCTTGGCCTTACCTGGATCCATGGGGAGTTCAGAATCCTGAAGTTT TCATTGAATCTTTTCATCAGGGTGAGAAAATTCTGATCTTTATAATCAAATCCTTTCTG GAAAACAACGGAGCAGATCACATTGCAGGGAGCACAGCCCAGGATGAAAGTGGGAT CACAGGGTGAAGCTAAAGATTTAAAAATTTTTAAAAAAATTATTAAAAAATAAATATT TAAAAGATTTGCATTTGTTAAGACATAAAGGAAATTTAGAAATTTTAAACAATATCTT ACAAATTCCCCATGTGTCCAAA Nucleic acidsequences are shown 5′-3′. DNA is uppercase. The location of the SNP isunderlined.

The results are shown in FIGS. 5A and 5B. From this, it is clear thatthe universal rhPCR genotyping system can be used to characterizemulti-allelic genotypes.

A Tri-allelic AD 360plot was designed for illustrating allelicdiscrimination. Fluorescence signal (ΔRn) from the last PCR cycle ofeach dye was normalized across the three dyes from the same well. Angleand distance of data point from the origin is calculated using formulabelow:

${Angle} = {{\tan^{- 1}\left( {\Delta \; {{RnDye}_{1} \div \Delta}\; {RnDye}_{2}} \right)} \times \frac{120}{90}}$${{Distance}\mspace{14mu} {from}\mspace{14mu} {origin}} = \sqrt{\left( {\Delta \; {RnDye}_{1}} \right)^{2} + \left( {\Delta \; {RnDye}_{2}} \right)^{2}}$

FIG. 5B shows the Tri-allelic Allelic Discrimination 360plot ofrs72558195, using rhPCR genotyping assay with 3 allele-specific primersmultiplexed in a single reaction. By collecting fluorescence signal fromall assays, six genotypes could be detected in a single reaction. Thedistance of data points from origin indicated the signal strength ofdyes and the wide angle separation between data clusters indicatedspecificity of multiplex assay. NTC in the center of the plot indicatedno primer dimers or non-specific amplification. The specificity ofmultiplex assay is achieved by the selectivity of RNase H2 and themutant Taq DNA polymerase as used in the previous examples. This AD360plot will also enable auto-calling capability by genotyping software.

A 360plot could be implemented for tetra-allelic, penta-allelic orhexa-allelic visualization. Therefore, visualization is possible forpositions that could have multiple bases as well as potential deletions.The distance from origin remains unchanged for each calculation, and theangle formulas would be:

  tetra-allelic  (4  alleles):  Angle = tan⁻¹(Δ RnDye₁ ÷ Δ RnDye₂)${{penta}\text{-}{a{llelic}}\mspace{11mu} \left( {5\mspace{14mu} {alleles}} \right)\text{:}\mspace{14mu} {Angle}} = {{\tan^{- 1}\left( {\Delta \; {{RnDye}_{1} \div \Delta}\; {RnDye}_{2}} \right)} \times \frac{72}{90}}$$\mspace{20mu} {{{hexa}\text{-}{allelic}\mspace{11mu} \left( {6\mspace{14mu} {alleles}} \right)\text{:}\mspace{14mu} {Angle}} = {{\tan^{- 1}\left( {\Delta \; {{RnDye}_{1} \div \Delta}\; {RnDye}_{2}} \right)} \times \frac{60}{90}}}$

Example 8

The following example illustrates the capability of the methods of thepresent invention to provide quantitative SNP genotyping, allowing fordetermination of the copy numbers of different alleles. To demonstratethis, an assay was designed against rs1135840, a SNP in the human CYP2D6gene. This gene can be present in multiple copies, and the number ofcopies with the rs1135840 SNP appears to affect drug metabolism (rapidmetabolism of the drug Debrisoquine).

To demonstrate that the assay system can detect small differences inallele rations, a standard curve for analysis was created. Two gBlock™(IDT, Coralville, Iowa) gene fragments were synthesized (Allele 1 andAllele 2, representing the two allelic variants (G>C) of the rs1135840SNP) and then mixed at different ratios. Reactions were performed in 10μL volumes, containing a total of 1500 copies of template at the ratiosshown (10:0, 9:1, 8:2, 7:3, 6:4, 5:5, 4:6, 3:7, 2:8, 1:9, and 0:10), 250nM of universal FAM probe (SEQ ID NO: 14), 450 nM of universal YakimaYellow® (SEQ ID NO: 22) probe, 1000 nM of universal forward primer (SEQID NO: 21), 150 nM of the two allele-specific forward primers, 500 nM ofthe reverse primer, and 5 μL of 2× Integrated DNA Technologies (IDT)(Coralville, Iowa) rhPCR genotyping master mix (containing dNTPs, amutant H784Q Taq polymerase (see Behlke, et al. U.S. 2015/0191707),chemically modified Pyrococcus abyssi RNase H2 (See Walder et al.UA20130288245A1), stabilizers, and MgCl₂).

PCR was performed on Life Technologies (Carlsbad, Calif.) QuantStudio™ 7Flex real-time PCR instrument using the following cycling conditions: 10mins at 95° C. followed by 45 cycles of 95° C. for 10 seconds and 60° C.for 45 seconds. End-point analysis of each of the plates was performedafter 45 cycles with software provided by the respective companies(Bio-Rad CFX Manager 3.1 software (Bio-Rad, Hercules, Calif.) andQuantStudio™ Real-Time PCR Software v1.3 (Carlsbad, Calif.)).

TABLE 16 Oligonucleotide sequences used in Example 8. SEQ Name SequenceID NO. Universal CGGCCCATGTCCCAGCGAA SEQ Forward ID NO. 21 primer Probe1 FAM-C+CATC+A+C+CGTG+CT-IBFQ SEQ ID (FAM) NO. 14 Probe 2Yak-CAATC+C+C+CGAG+CT-IBFQ SEQ ID (Yakima NO. 22 Yellow) rs1135840GCCCATGTCCCAGCGAACCATCACCGTGC SEQ ID Allele 1TGTCTTTGCTTTCCTGGTGAGcCCATG-x NO. 38 Forward primer rs1135840GCCCATGTCCCAGCGAACAATCCCCGAGC SEQ ID Allele 2TGTCTTTGCTTTCCTGGTGAcCCATG-x NO. 39 Forward primer rs1135840GCGTTGGAACTACCACATTGCTTTATuGTA SEQ ID Reverse CT-x NO. 40 primer Nucleicacid sequences are shown 5′-3′. DNA is uppercase, RNA is lowercase. LNAresidues are designated with a +. Location of potential mismatch isunderlined. FAM = 6-carboxyfluorescein, Yak = Yakima Yellow(3-(5,6,4′,7′-tetrachloro-5′-methyl-3′,6′-dipivaloylfluorescein-2-yl)),IBFQ = Iowa Black FQ (fluorescence quencher), and x = C3 propanediolspacer block.

The resulting data is illustrated in FIG. 7. The spread of each of thesample mixes is sufficient for the determination of the number of copiesof each template.

After demonstration of the required amount of separation of allelicquantities, it is possible to determine the number of copies present ofeach allele in an experimental sample. To test this, the previouslydescribed assay designed against rs1135840, was utilized to testthirteen Coriell genomic DNA (Camden, N.J.) samples with varying CYP2D6copy numbers with varying rs1135840 genotypes. These samples have knowndefined copy numbers and rs1135840 genotypes which could be verifiedafter testing with the universal rhPCR genotyping mix. From this, thesesamples can also be categorized as being homozygotes for either allele,or heterozygotes.

To calculate the copy number from the data, two duplex reactions wererun for each sample. Reactions were performed in 10 μL volumes,containing 3 ng of one of the following genomic DNAs: NA17123, NA17131,NA17132, NA17149, NA17104, NA17113, NA17144, NA17213, NA17221, NA17114,NA17235, or NA17241. Each individual assay also contained 50 nM ROXnormalizer oligo, 250 nM of universal FAM probe (SEQ ID NO: 14), 450 nMof universal Yakima Yellow® (SEQ ID NO: 22) probe, 1000 nM of universalforward primer (SEQ ID NO: 21), 150 nM of the two allele-specificforward primers (SEQ ID NO: 38 and 39), 500 nM of the reverse primer(SEQ ID NO: 40), and 5 μL of 2× Integrated DNA Technologies (IDT)(Coralville, Iowa) rhPCR genotyping master mix (containing dNTPs, amutant H784Q Taq polymerase (see Behlke, et al. U.S. 2015/0191707),chemically modified Pyrococcus abyssi RNase H2 (See Walder et al.UA20130288245A1), stabilizers, and MgCl₂). Assays also contained aseparate RNase P assay (See table 17, SEQ ID NOs: 41-43)) fornormalization of the template concentration.

TABLE 17 RNase P assay sequences used in Example 8. Name Sequence SEQ IDNO. RNase P GCGGAGGGAAGCTCATCAG SEQ ID NO. 41 Forward primer RNase PCCCTAGTCTCAGACCTTCCCAA SEQ ID NO. 42 Reverse primer Probe 2Yak-CCACGAGCTGAGTGCGTCCTGTCA- SEQ ID NO. 43 (Yakima IBFQ Yellow) Nucleicacid sequences are shown 5′-3′. DNA is uppercase. FAM= 6-carboxyfluorescein, Yak = Yakima Yellow(3-(5,6,4′,7′-tetrachloro-5′-methyl-3′,6′-dipivaloylfluorescein-2-yl)),IBFQ = Iowa Black FQ (fluorescence quencher).

Quantitative PCR was performed on Life Technologies (Carlsbad, Calif.)QuantStudio™ 7 Flex real-time PCR instrument using the following cyclingconditions: 10 mins at 95° C. followed by 45 cycles of 95° C. for 10seconds and 60° C. for 45 seconds. End-point analysis of each of theplates was performed after 45 cycles with the QuantStudio™ Real-Time PCRSoftware v1.3 (Carlsbad, Calif.) software provided by the company.

Copy number was determined by the following method. For each sampleshown to be a homozygote, ΔCq (RNase P Cq—rs1135840 assay Cq) wascalculated for each sample. For samples shown to be heterozygotes, ΔCqwas calculated for both alleles (RNase P Cq—rs1135840 assay 1 Cq andRNase P Cq—rs1135840 assay 2 Cq). Next, ΔΔCq (ΔCq—mean ΔCq for known 2copy control DNA samples) was calculated for each allele. Thiscorrection allowed for normalization against amplification differencesbetween the SNP assay and the RNase P assay. Finally, the followingequation was used to calculate copy number for each allele:

Copy number of allele=2*(2̂(ΔΔCq))

The resulting end-point data is shown in FIG. 8A and calculated copynumbers are shown in FIG. 8B. The genotypes determined in FIG. 8A(homozygotes allele 1, Homozygotes allele 2, or heterozygote) allmatched the known genotypes, and allowed correct calculation of the copynumber. The established reference copy number of the individual samplesis shown under each result. In each case, the copy number determined bythe assay correctly determined the genotype and copy number of the inputDNA.

Example 9

The following example demonstrates that a variation of an rhPCR probecan be used for multiplexed rhPCR.

The assay schematic is provided in FIG. 9. In the first round of PCR, 5′tailed target-specific rhPrimers are used. The 5′ tails uponincorporation into the amplicon contain binding sites for a second roundof PCR with different primers (blocked or unblocked) to add applicationspecific sequences. For example, as depicted in FIG. 9, this system canbe used for amplification enrichment for next generation sequencing. Inthis case, 5′ tailed rhPCR primers contain read 1/read 2 primersequences. The second round of PCR adds adapter sequences such as theP5/P7 series for Illumina® based sequencing platforms or other adaptors,including ones containing barcodes/unique molecular identifiers. Thisapproach allows for adding any additional sequences onto the ampliconnecessary for input into any NGS platform type.

As illustrated in FIG. 10, two primers sets, including one containing a96-plex set of 5′ tailed rhPrimers, and one containing 96 DNA “standard”5′ tailed PCR primers were designed using an IDT algorithm. The twoprimer sets differed only in that the rhPrimers contained an internalcleavable RNA base and a blocking group on the 3′ end. Once the blockinggroup was removed by RNase H2 cleavage, the primer sequences becomeidentical.

The first round of PCR reactions contained the 96 plex at 10 nM of eachblocked target specific primer, 10 ng of NA12878 human genomic DNA(Coriell Institute for Medical Research, Camden, N.J.), 200 mU ofchemically modified Pyrococcus abyssi RNase H2 (See Walder et al.UA20130288245A1) (IDT, Coralville, Iowa) and 1×KAPA 2G HotStart FastReady Mix™ (Kapa Biosystems, Wilmington, Mass.). The thermal cyclingprofile was 10 mins at 95° C. followed by 8 cycles of 95° C. for 15seconds and 60° C. for 4 minutes, and a final 99° C. finishing step for15 minutes. Reactions were cleaned up with a 2×AMPure™ XP beads (BeckmanCoulter, Brea, Calif.). Briefly, 100 μL AMPure™ SPRI beads were added toeach PCR well, incubated for 5 minutes at room temperature and collectedfor 5 minutes at room temperature on plate magnet (DynaMag™(Thermo-Fisher, (Watherham, Mass.) 96-well plate side-magnet). Beadswere washed twice with 80% ethanol, and allowed to dry for 3 minutes atroom temperature. Samples were eluted in 22 μL of TE at pH 8.0.

The second round of PCR was set up using 20 μL of the cleaned up firstround PCR products, universal PCR-50F and PCR-47R primers (See table 18,SEQ ID NOs: 44 and 45) at 2 uM and 1×KAPA 2G HotStart Fast Ready Mix™(KAPA Biosystems, Wilmington, Mass.). Reactions were cycled for 45seconds at 98° C. followed by 20 cycles of 98° C. for 15 seconds, 60° C.for 30 seconds, and 72° C. for 30 seconds. A final 1 minute 72° C.polishing step finished the reaction. Samples were cleaned up again with0.8×AMPure™ beads. Briefly, 40 μL AMPure™ SPRI beads were added thesecond PCR wells, incubated for 5 minutes at room temperature andcollected for 5 minutes at room temperature on plate magnet (DynaMag™(Thermo-Fisher, (Watherham, Mass.) 96-well plate side-magnet). Beadswere washed twice with 80% ethanol, and allowed to dry for 3 minutes atroom temperature. Samples were eluted in 22 μL of TE at pH 8.0, and 20μL was transferred to a new tube.

2 μL of the samples were analyzed using the Agilent® High SensitivityD1000™ Screen Tape™ on the Agilent® 2200 Tape Station™ (AgilentTechnologies®, Santa Clara, Calif.). Quantification was performed usingthe KAPA Library Quantification Kit (KAPA Biosystems, Wilmington, Mass.)for Illumina® Platforms, according to the manufacturer's protocol.Replicate samples were pooled to a final concentration of 10 pM, and 1%PhiX bacteriophage sequencing control was added. Samples were run with aV2 300 cycle MiSeq™ kit on an Illumina® (San Diego, Calif.) MiSeq™platform, using standard protocols from the manufacturer.

TABLE 18 Universal assay sequences used in Example 9. SEQ Name SequenceID NO. Universal AATGATACGGCGACCACCGAGATCTACAC SEQ ID NO. PCR-50FTCTTTCCCTACACGACGCTCT 44 Universal CAAGCAGAAGACGGCATACGAGATGGACC SEQ IDNO. PCR-47R TATGTGACTGGAGTTCAGACGTGTGC 45 Nucleic acid sequences areshown 5′-3′. DNA is uppercase.

FIG. 10 shows the results from the Agilent® Tape Station. The primerdimer product was the most significant product produced using standardDNA primers in the presence of DNA template, with only a small amount offull length expected product. In the absence of template, the primerdimer product was the major component of the reaction. In the case ofthe blocked rhPCR primers, the vast majority of the material was thedesired PCR products, with little primer dimer observed. In the absenceof template, there is no primer dimer present, contrasting with theoverwhelming abundance of primer dimer observed in the no template laneof the unblocked DNA primers. Quantitation of the product versus primerdimer bands show that mass ratio of product to primer dimer for theunblocked DNA primers was 0.6. The mass ratio for the rhPCR primers was6.3.

FIG. 11 summarizes two key sequencing metrics. The first is the percentof mapped reads from the sequencing data. The rhPCR reactions gave apercentage of reads mapped to the human genome at 85%, whereas thenon-blocked DNA primers on give a mapped read percentage of less than20. A second metric, the percentage of on-target reads, is almost 95%when using rhPCR primers, but less than 85% when the non-blocked primersare used in the multiplex. These results clearly demonstrate the utilityof using rhPCR in multiplexing, where a large increase of the desiredmaterial is seen, and a vast reduction in undesired side products isobserved. The differences mean less unwanted sequencing reads, and thedepth of coverage of desired sequences is higher.

Example 10

This example demonstrates enhanced sensitivity and accuracy of assaysystems of the disclosure as compared to standard T7 endonucleasecleavage assays.

A total of 36 sites modified with the CRISPR/Cas9 protein system werechosen to be comparatively analyzed by T7 endonuclease, next-generationsequencing (NGS), or a qPCR “Genie” assay system of the disclosure.

To mutate the chosen genomic sites, a HEK293 cell line was generatedwith stable expression of S pyogenes Cas9. AltR™ guide RNAs were reversetransfected into the cells in 96-well plates (40,000 cells/well) using0.75 μL RNAiMAX (Thermo) per well. Briefly, AltR™ crRNAs were designedfrom the IDT CRISPR2.0 design engine to target exon 1 or 2 of selectedhuman genes. Prior to transfection, guide RNAs (crRNA/tracrRNA withAltR™ chemistry) were duplexed in an equimolar ratio at 3 μM finalconcentration of the complex in IDT duplex buffer (Integrated DNATechnologies, Coralville, Iowa). Complexes were heated to 95° C. for 5min and cooled to room temperature. Genomic DNA was isolated after 48hrs with 50 μL Quick Extract buffer (Epicentre), using standardtechniques described by the manufacturer. DNA solutions were furtherdiluted with 100 μL water before further analysis was performed.

Analysis by T7 endonuclease digestion was done as follows.CRISPR-Cas9-treated cells were washed with 100 μL of PBS. Cells werelysed by adding 50 μL of QuickExtract™ DNA Extraction Solution(Integrated DNA Technologies). Cell lysates were then transferred toappropriate PCR tubes or plate, then vortexed and heated in a thermalcycler at 65° C. for 10 min, followed by 98° C. for 5 min, after which100 μL of Nuclease-Free Water was added to dilute the genomic DNA. Thesamples were then vortexed and spun down. PCR was set up using template,primers, and components of the Alt-R Genome Editing Detection Kit andKAPA HiFi HotStart PCR Kit as follows. Sample: 4 μL (˜40 ng) genomicDNA, 300 nM forward primer, 300 nM reverse primer, 5 μL (1λ) of KAPAHiFi Fidelity Buffer (5λ), 1.2 mM (0.3 mM each) dNTPs, 0.5 U KAPA HiFiHotstart DNA Polymerase (1 U/μL), for a total volume of 25 μL. Alt-R™Control A: 1 μL Alt-R™ Control A template/primer mix, 5 μL (1×) of KAPAHiFi Fidelity Buffer (5×), 1.2 mM (0.3 mM each) dNTPs, 0.5 U KAPA HiFiHotstart DNA Polymerase (1 U/μL), for a total volume of 25 μL. Alt-R™Control B: 1 μL Alt-R™ Control B template/primer mix, 5 μL (1×) of KAPAHiFi Fidelity Buffer (5×), 1.2 mM (0.3 mM each) dNTPs, 0.5 U KAPA HiFiHotstart DNA Polymerase (1 U/μL), for a total volume of 25 μL. PCR wasrun using the following conditions: denature at 95° C. for 5 min; 30cycles of: denature at 98° C. for 20 sec, anneal between 64-67° C.(depending on polymerase) for 15 sec, extend at 72° C. for 30 sec; thenextend at 72° C. for 2 minutes. Heteroduplexes for T7EI digestion wereformed as follows. 2 μL T7EI Reaction Buffer (10×) and 6 μLNuclease-Free Water was combined with 10 μL experimental target or Alt-RHPRT control from the PCR, 10 μL Control A PCR component (homoduplexcontrol), or 5 μL Control A and 5 μL Control B (heteroduplex control).The PCR products were then placed in a thermal cycler with 95° C.denaturation for 10 min, ramp from 95-85° C. at a ramp rate of −2°C./sec, then ramp from 85-25° C. at a ramp rate of −0.3° C./sec. 18 μLof PCR heteroduplexes from the previous step were combined with 2 μL T7endonuclease I (1 U/μL), then the T7EI reaction was incubated at 37° C.for 60 min. T7EI mismatch detection result were visualized on a FragmentAnalyzer™ system with Mutation Discovery Kit according to themanufacturer's instructions (Integrated DNA Technologies). Afteramplification and cleavage, amplicons were sized on the FragmentAnalyzer™ (Advanced Analytical, Inc, Ames, Iowa) capillaryelectrophoresis system.

NGS sequencing analysis of the mutated samples employed locus-specificprimers positioned approximately 75-bp flanking the Cas9 cleavage site.Primers contained universal 5′-tails that allowed for secondaryamplification that added Illumina™ TruSeq™ i5 and i7 adapters withsample-specific barcodes to the amplicons. The locus-specific primerswere designed as RNase H2-cleavable primers with the 4DMX blockingmodification at the 3′-end (where the 4DMX nomenclature indicates 4 DNAbases, a mismatched DNA base and a propanediol C3-spacer 3′ of the RNAbase). Using a master-mix containing a hot-start Taq polymerase andhot-start RNaseH2, the genomic DNAs were amplified using the followingcycling conditions: 95° C.^(5:00)+(95° C.^(0:15)+60° C.^(1:00))×8cycles+99° C.^(15:00). Samples were purified using SPRI beads (1.5×Agencourt™ Ampure® XP beads, Beckman Coulter) per the manufacturer'sprotocol. The second PCR incorporated the Illumina adapters and was rununder the following conditions: 95° C.^(5:00)+(95° C.^(0:15)+60°C.^(0:30)+72° C.^(0:30))×18 cycles+99° C.^(15:00).

The resultant amplicons underwent 1×SPRI™ clean-up and were quantifiedvia the KAPA™ library qPCR quantitation (KAPA Biosystems, Wilmington,Mass.) kit per the manufacturer's recommended protocol. In addition,amplicons were sized on the Fragment Analyzer™ (Advanced Analytical,Inc., Ames, Iowa) capillary electrophoresis system.

DNA sequencing was carried out on an Illumina™ MiSeq® using a MiSeq®Nano cartridge (v2, 300 cycles). Data was de-multiplexed via an in-housebioinformatics processing pipeline. Analysis for specific editing eventsrelative to a reference amplicon was performed with CRISPResso™ usingmethods described.

Quantitative PCR assay primers for analysis according to methods of thedisclosure were designed so that the RNA nucleotide was located twobases after the primary Cas9 cleavage site, allowing for maximaldiscrimination from both the RNase H2 enzyme and the DNA polymerase(FIG. 12). Primers were designed to include a proprietary universal 5′domain (UniFor-UniPro-), which has sequence identity with both auniversal forward primer, and a universal 5′ nuclease degradable probe(Table 1, Seq ID No. 1-72 and Table 2, Seq ID No. 76-147). ATaqman-based RNase P assay (Seq ID No. 73-75) was utilized as auniversal control for template concentration normalization in all cases.All primers were synthesized at Integrated DNA Technologies (IDT,Coralville, Iowa). Amplification was performed with 2.5 μL of the sameQuickExtract™ genomic DNA utilized in T7 and NGS analyses. A wild-type(WT) control that was grown and extracted by the same method was alsoanalyzed for normalization purposes. Reaction volumes were 10 μL in allcases, and included the universal forward primer, the universal probe,the interrogating primer, and the non-interrogating reverse primer. 1xof a rhPCR-genotyping master mix, containing hot-start RNase H2, athermophilic DNA polymerase, buffer, and dNTPs was also included. Theuniversal forward primer was present in all reactions at 1000 nM (10pmol), and the universal assay probe was present at 300 nM (3 pmol). Theassay specific primers were present at 200 nM (2 pmol) for the forward(mutation interrogating) primer, while the non-interrogatinglocus-specific reverse primer was present at 500 nM (5 pmol). RNase Pcontrol reactions were run with 500 nM of forward and reverse RNase Pprimers, and 250 nM probe (Seq ID No. 73-75). Reactions were run on aCFX384™ Real-Time qPCR machine (Bio-Rad®, Hercules, Calif.). Thefollowing cycling conditions were utilized: 95° C.^(10:00)+(95°C.^(0:15)+59° C.^(0:20)+72° C.^(0:30))×55 cycles.

TABLE 1 Discriminatory forward primers utilized in Example 1. Seq IDName Sequence No. GCK-356-1 UniFor-UniPro-CCCTGGGTCCCTGGGaGAATC-x 46GCK-356-2 UniFor-UniPro-CGAGGAGAACCACATTCTCCcAGGGT-x 47 ERBB3-33-1UniFor-UniPro-GGGCGGCCGTGACuCACC-x 48 ERBB3-33-2UniFor-UniPro-GAGGGAAGGGGGTGAGTcACGGCG-x 49 TTR-1257-1UniFor-UniPro-CCTGGGAGCCATTTGCCTCuGGGTT-x 50 TTR-1257-2UniFor-UniPro-CTTTGGCAACTTACCCAGAGGcAAATC-x 51 HAMP-253-1UniFor-UniPro-GCACTGAGCTCCCAGAuCTGGC-x 52 HAMP-253-2UniFor-UniPro-GCAAGCGGCCCAGATCuGGGAC-x 53 BIRC5-606-1UniFor-UniPro-GACGACCCCATGTAAGTCTTCuCTGGG-x 54 BIRC5-606-2UniFor-UniPro-CGAGGCTGGCCAGAGAaGACTTT-x 55 SAA 146-1 UniFor-UniPro- 56CTTTCCCAACAAGATTATCATTTCCTTTAAaAAAAT-x SAA 146-2UniFor-UniPro-CGCCCCAGGATAACTATTTTTTTTaAAGGT-x 57 IDO1-97-1UniFor-UniPro-AGACACTGAGGGGCACCaGAGGT-x 58 IDO1-97-2UniFor-UniPro-CTTGTAGTCTGCTCCTCTGGuGCCCG-x 59 IDO1-176-1 UniFor-UniPro-60 AGTAAAGAGTACCATATTGATGAAGAAgTGGGA-x IDO1-176-2UniFor-UniPro-GCAGAGCAAAGCCCACTTcTTCAA-x 61 CYP27A-UniFor-UniPro-CCTTTGGTGAGGACTCCCAgATGGC-x 62 31016-1 CYP27A-UniFor-UniPro-CCTGGGCCCCATCTGgGAGTG-x 63 31016-2 SAA 226-1UniFor-UniPro-TCTCCTCTGATCTAGAGAGGTAAGcAGGGA-x 64 SAA 226-2UniFor-UniPro-ACCAGGCCCGACCCTGCTuACCTG-x 65 KIF11-369-1UniFor-UniPro-GAGAAGGGGAAGAACATCCAgGTGGA-x 66 KIF11-369-2UniFor-UniPro-GCATCTCACCACCACCTGgATGTA-x 67 C3-1394-1UniFor-UniPro-CTGGACAGCACTAGTTTTTTGCcTGGGT-x 68 C3-1394-2UniFor-UniPro-CCACGACTTCCCAGGCaAAAAT-x 69 HOGA-505-1UniFor-UniPro-CACTGCAGAGGTGGACTaTGGGT-x 70 HOGA-505-2UniFor-UniPro-GATTCTCCTCCAGTTTCCCATAGuCCACG-x 71 EGFR-UniFor-UniPro-CCAGAGGATGTTCAATAACTGTGAgGTGGA-x 72 123344-1 EGFR-UniFor-UniPro-CAAATTCCCAAGGACCACCuCACAC-x 73 123344-2 ALDH2-UniFor-UniPro-TGAAGGGGACAAGGTGAGAaCTGGA-x 74 15144-1 ALDH2-UniFor-UniPro-CCCAAGGTAAGTCACCAGTTCuCACCA-x 75 15144-2 AGXT-140-1UniFor-UniPro-CCATGGCCTCTCACAAGCTgCTGGA-x 76 AGXT-140-2UniFor-UniPro-GGGGGTCACCAGCAGcTTGTC-x 77 APOC-2929-1UniFor-UniPro-CCGTTAAGGACAAGTTCTCTGAGTuCTGGC-x 78 APOC-2929-2UniFor-UniPro-TCAGGGTCCAAATCCCAGAACuCAGAC-x 79 Met 27554-1UniFor-UniPro- 80 AATTTTATTTACTTCTTGACGGTCCAAAGGgAAACA-x Met 27554-2UniFor-UniPro-GTCTGAGCATCTAGAGTTTCCCuTTGGT-x 81 SAA 88-1UniFor-UniPro-AGGTGAGGAGCACACCAAGGAgTGATA-x 82 SAA 88-2 UniFor-UniPro-83 GAAAACAGAGTAAGTTTTAAAAATCACTCcTTGGA-x HIF1A-293-1UniFor-UniPro-TCGCACCCCCACCTcTGGAG-x 84 HIF1A-293-2UniFor-UniPro-GAAGGAAAGGCAAGTCCAGAGgTGGGC-x 85 Met 27475-1UniFor-UniPro- 86 AATTTTATTTACTTCTTGACGGTCCAAAGGgAAACA-x Met 27475-2UniFor-UniPro-GTCTGAGCATCTAGAGTTTCCCuTTGGT-x 87 HAMP-295-1UniFor-UniPro-CTCGCCAGCCTGACCaGTGGG-x 88 HAMP-295-2UniFor-UniPro-GGGAAAACAGAGCCACTGGuCAGGG-x 89 GRHPR-UniFor-UniPro-GCCTCCTCTCCGACCAcGTGGT-x 90 2234-1 GRHPR-UniFor-UniPro-GGATCCTCTTGTCCACGTGgTCGGAC-x 91 2234-2 HAMP-88-1UniFor-UniPro-GGCGCCACCACCTTcTTGGT-x 92 HAMP-88-2UniFor-UniPro-GCTCTGTCTCATTTCCAAGAAgGTGGA-x 93 Met 27254-1UniFor-UniPro-GAGCCAAAGTCCTTTCATCTGTaAAGGT-x 94 Met 27254-2UniFor-UniPro-GAAGTTGATGAACCGGTCCTTTACaGATGT-x 95 GRHPR-UniFor-UniPro-ACAAGAGGATCCTGGATGCTgCAGGA-x 96 2264-1 GRHPR-UniFor-UniPro-CGCTCTAGCTCCTTGGCaGGGAA-x 97 2264-2 Serpina 279-1UniFor-UniPro-ACTCAGTTCCACAGGTGGGAGgGAGGC-x 98 Serpina 279-2UniFor-UniPro-CACTCTAAGCCCTGCTGTCCCaCCTGA-x 99 Myc 459-1UniFor-UniPro-CGGGAGGCTATTCTGCCCATTuGGGAT-x 100 Myc 459-2UniFor-UniPro-CGGGGAAGTGTCCCCAAAuGGGCT-x 101 Serpina 130-1UniFor-UniPro-GCTGCTGCTGCCAGGAAuTCCAC-x 102 Serpina 130-2UniFor-UniPro-CCCCTCCAACCTGGAATTcCTGGG-x 103 Myc 490-1UniFor-UniPro-CTGCCAGGACCCGCTTCuCTGAT-x 104 Myc 490-2UniFor-UniPro-CAAGGAGAGCCTTTCAGAGAaGCGGC-x 105 GRHPR-UniFor-UniPro-GATGAGCCCATCCCTGCcAAGGT-x 106 2179-1 GRHPR-UniFor-UniPro-CGCTCTAGCTCCTTGGCaGGGAA-x 107 2179-2 GYG-2851-1UniFor-UniPro-TCGCCACCCCTCAGGuCTCAC-x 108 GYG-2851-2UniFor-UniPro-ACCTCATGGAGTCTGAGACCuGAGGC-x 109 GYG-2793-1UniFor-UniPro-GCCCTGGTCCTGGGAuCATCA-x 110 GYG-2793-2UniFor-UniPro-CTGTGCTGTTTCAGAGATGATCcCAGGT-x 111 Serpina 79-1UniFor-UniPro-CAAGAGTCCTGAGCTGAACCAAgAAGGT-x 112 Serpina 79-2UniFor-UniPro-CGACCCCCTCCTCCTTCTTgGTTCT-x 113 Myc 538-1UniFor-UniPro-CTGCTTAGACGCTGGATTTTTTuCGGGA-x 114 Myc 538-2UniFor-UniPro-CTGGTTTTCCACTACCCGAAArAAAAA-x 115 GYG-2744-1UniFor-UniPro-CTTTGTATTAAGATCAGGCCTTTGTgACACA-x 116 GYG-2744-2UniFor-UniPro-CATCGTTTGTGGTTAGTGTCACaAAGGG-x 117 RNase P ForGCGGAGGGAAGCTCATCAG 118 RNase P Rev CCCTAGTCTCAGACCTTCCCAA 119 RNase PFAM-CCACGAGCTGAGTGCGTCCTGTCA-IBFQ 120 probe DNA is uppercase, RNA islowercase. FAM = 6-Fluorescein fluorescent dye (IDT, Coralville, IA).IBFQ = Iowa Black ™ fluorescent quencher (IDT, Coralville, IA).UniFor-UniPro = universal forward primer, and universal probe bindingsite. X = propanediol (C3) spacer blocking group.

TABLE 2 Non-discriminatory reverse primers utilized in Example 1. SeqName Sequence ID No. GCK-356-1 GAGGAAACTGTGACTGAACCTC 121 GCK-356-2CCAAGGCTTCTCCGCC 122 ERBB3-33-1 GAGTCCGGGGAGGGATG 123 ERBB3-33-2CAATCCCTACTCCAGCCTC 124 TTR-1257-1 ATGTGAGCCTCTCTCTACCAA 125 TTR-1257-2GTCCTCTGATGGTCAAAGTTCTA 126 HAMP-253-1 CACTGGTCAGGCTGGC 127 HAMP-253-2CAAGCTCAAGACCCAGCA 128 BIRC5-606-1 CAACTCAAATCTTTTGACAACTCAG 129BIRC5-606-2 GGAGCTGGAAGGCTGG 130 SAA 146-1 TTCAGAATGGTATGGCTGTATGC 131SAA 146-2 CACAGATCAGGTGAGGAGCA 132 IDO1-97-1 GTTTTCCATAGCGTGTGCC 133IDO1-97-2 GTGGTCACTGGCTGTGG 134 IDO1-176-1 TTCCCACATTTTACTGCCTTCTC 135IDO1-176-2 CGCTATGGAAAACTCCTGGA 136 CYP27A- CAGGTCTGTGCATCAGCG 13731016-1 CYP27A- CTTTCTGGAAGCGATACCTG 138 31016-2 SAA 226-1CGCACAGAACTCAACATGGG 139 SAA 226-2 AATAGTTATCCTGGGGCATACAGC 140KIF11-369-1 GCTCGGAATCCTGTCAGC 141 KIF11-369-2 CAGCCAAATTCGTCTGCG 142C3-1394-1 GGGATGTTCCAGTCACTGTTAC 143 C3-1394-2 GGTTGGTGGCAGGGG 144HOGA-505-1 AGGGGAAGGTGCCCAG 145 HOGA-505-2 AAGGTGGACATTGCGGG 146 EGFR-TCATAATTCCTCTGCACATAGGT 147 123344-1 EGFR- GCCAAGGCACGAGTAACA 148123344-2 ALDH2- CGTATAAAATAGAAGACGAATCCATCCC 149 15144-1 ALDH2-ATGGCACGATGCCGT 150 15144-2 AGXT-140-1 GGCTTGAGCAGGGCC 151 AGXT-140-2TGGCCAAGGCCAGTG 152 APOC-2929-1 TCAGGCAGCCACGGC 153 APOC-2929-2GTGACCGATGGCTTCAGT 154 Met 27554-1 CATACGCAGCCTGAAGTATATTAAACA 155 Met27554-2 TAGATGCTCAGACTTTTCACACAAGA 156 SAA 88-1 TTCAGAATGGTATGGCTGTATGC157 SAA 88-2 AGCAGGGAAGGCTCAGTATAAATAG 158 HIF1A-293-1 TAAGCGCTGGCTCCCT159 HIF1A-293-2 CTCTAGTCTCACGAGGGGTT 160 Met 27475-1CTCTTTTCTGTGAGAATACACTCCAG 161 Met 27475-2 TACCCCATTAAGTATGTCCATGCC 162HAMP-295-1 TCTCCCATCCCTGCTGC 163 HAMP-295-2 CCGCTTGCCTCCTGC 164 GRHPR-CACCCAGTGTGCACCT 165 2234-1 GRHPR- GCCAAGGAGCTAGAGCGA 166 2234-2HAMP-88-1 GAGGCGGTGGTCTGAG 167 HAMP-88-2 TGTTCCCTGTCGCTCTG 168 Met27254-1 CTTTAGCCTTCTCACTGATATCGAATG 169 Met 27254-2GCATATTCTCCCCACAGATAGAAGA 170 GRHPR- CCTGCCCACCCAGTG 171 2264-1 GRHPR-CTGTGAGGTGGAGCAGTG 172 2264-2 Serpina 279-1 TAGCTCCTGGGCATTTCTTCC 173Serpina 279-2 AGCTTGAGGAGAGCAGGAAAG 174 Myc 459-1 CCTGGTTTTCCACTACCCGA175 Myc 459-2 CACTGGAACTTACAACACCCG 176 Serpina 130-1TTCCTGCTCTCCTCAAGCTCT 177 Serpina 130-2 GAGCTGAACCAAGAAGGAGGA 178 Myc490-1 AGGCATTCGACTCATCTCAGC 179 Myc 490-2 TGCACTGGAACTTACAACACC 180GRHPR- TCGGAGAGGAGGCAGAG 181 2179-1 GRHPR- TTCTCCTGAGGGCCTCC 182 2179-2GYG-2851-1 ACAGGGAGAAGGATGTCAGAG 183 GYG-2851-2 GTCCTGGGATCATCTCTGAAAC184 GYG-2793-1 ACAGGGAGAAGGATGTCAGAG 185 GYG-2793-2CACTAACCACAAACGATGCCT 186 Serpina 79-1 GAATTCCTGGCAGCAGCA 187 Serpina79-2 CTACTGCCTCCACCCGAA 188 Myc 538-1 TAGGCATTCGACTCATCTCAGC 189 Myc538-2 TGCACTGGAACTTACAACACC 190 GYG-2744-1 GGACCAGGGCACCTTTG 191GYG-2744-2 GGCTTTCTCCAGATAAGATACTG 192 DNA is uppercase.

Although the reverse primers were not cleaved by RNase H2 in theseassays, the results suggest that blocked-cleavable reverse primers couldalso be utilized in these assays.

Analysis of the amplification data was performed using a ΔΔCq method.Briefly, the ΔCq was calculated between each of the target Cqs and thecorresponding reference (RNase P) Cqs. A conversion was then done withthe calculated ΔCq, where ΔCq experimental was calculated as being equalto 2̂−ΔCq. ΔΔCq was then calculated by normalization against the ΔCqcalculated from the WT (un-mutated) control.

Experimental results are shown in FIGS. 13A and 13B. FIG. 13A shows theclear difference between the NGS and T7 endonuclease cleavage data. Ofthe 72 assays tested, 64 of the T7EI results were >25% discordant intheir quantification of the amount of mutant template present comparedto the NGS gold standard. The error in T7EI quantification is expectedas EMCA assays usually underestimate genome editing rates, as discussedabove.

FIG. 13B shows the comparison between the NGS data and the method of thepresent invention. A total of 74 assays were tested in the new assayformat. Of these, 13 failed to amplify (17%), of which 10/13 (76%) ofthe failed assays showed sequence features that impair primer functionand could easily be removed from future testing by a design algorithm(G-quadraplexes, hairpins, etc.). Of the 61/74 assays that amplified,only 2/61 (3.3%) showed more than 25% divergence from the resultsobtained with the NGS experiment. These data, combined with the datafrom FIG. 13A, demonstrate the utility of the assays of the disclosurein providing a rapid, inexpensive PCR-based method to detect CRISPRgenome mutation events and how the accuracy of this method is muchsuperior to EMCA assays.

All references, including publications, patent applications, andpatents, cited herein are hereby incorporated by reference to the sameextent as if each reference were individually and specifically indicatedto be incorporated by reference and were set forth in its entiretyherein.

The use of the terms “a” and “an” and “the” and similar referents in thecontext of describing the invention (especially in the context of thefollowing claims) are to be construed to cover both the singular and theplural, unless otherwise indicated herein or clearly contradicted bycontext. The terms “comprising,” “having,” “including,” and “containing”are to be construed as open-ended terms (i.e., meaning “including, butnot limited to,”) unless otherwise noted. Recitation of ranges of valuesherein are merely intended to serve as a shorthand method of referringindividually to each separate value falling within the range, unlessotherwise indicated herein, and each separate value is incorporated intothe specification as if it were individually recited herein. All methodsdescribed herein can be performed in any suitable order unless otherwiseindicated herein or otherwise clearly contradicted by context. The useof any and all examples, or exemplary language (e.g., “such as”)provided herein, is intended merely to better illuminate the inventionand does not pose a limitation on the scope of the invention unlessotherwise claimed. No language in the specification should be construedas indicating any non-claimed element as essential to the practice ofthe invention.

Preferred embodiments of this invention are described herein, includingthe best mode known to the inventors for carrying out the invention.Variations of those preferred embodiments may become apparent to thoseof ordinary skill in the art upon reading the foregoing description. Theinventors expect skilled artisans to employ such variations asappropriate, and the inventors intend for the invention to be practicedotherwise than as specifically described herein. Accordingly, thisinvention includes all modifications and equivalents of the subjectmatter recited in the claims appended hereto as permitted by applicablelaw. Moreover, any combination of the above-described elements in allpossible variations thereof is encompassed by the invention unlessotherwise indicated herein or otherwise clearly contradicted by context.

What is claimed is:
 1. A blocked-cleavable primer for rhPCR, the primercomprising: 5′-A-B-C-D-E-3′ wherein A is optional and is a tailextension that is not complementary to a target; B is a sequence domainthat is complementary to a target; C is a discrimination domain; D is acleavage domain that, when hybridized to the target, is cleavable byRNase H2, and which comprises an RNA base; and E is a blocking domainthat prevents extension of the primer.
 2. The primer of claim 1, whereinthe RNA base is separated from the discrimination domain by one baseposition.
 3. The primer of claim 1, wherein the RNA base is within thediscrimination domain.
 4. The primer of claim 1, wherein the RNA base isadjacent to the discrimination domain.
 5. The primer of claim 1, whereinD is comprised of 1-3 RNA bases.
 6. The primer of claim 1, wherein thecleavage domain comprises one or more of the following moieties: a DNAresidue, an abasic residue, a modified nucleoside, or a modifiedphosphate internucleotide linkage.
 8. The primer of claim 1, wherein asequence flanking the cleavage site contains one or more internucleosidelinkages resistant to nuclease cleavage.
 8. The primer of claim 7,wherein the nuclease resistant linkage is a phosphorothioate.
 9. Theprimer of claim 5, wherein the 3′ oxygen atom of at least one of the RNAresidues is substituted with an amino group, thiol group, or a methylenegroup.
 10. The primer of claim 1, wherein the blocking group is attachedto the 3′-terminal nucleotide of the primer.
 11. The primer of claim 1,wherein A is comprised of a region that is identical to a universalforward primer and optionally a probe binding domain.
 12. The primer ofclaim 1, wherein the discrimination domain C comprises or overlaps withthe cleavage domain D.
 13. A method of detecting a variation in a targetDNA sequence that has been altered with a gene editing enzyme, themethod comprising: (a) providing a reaction mixture comprising: (i) anoligonucleotide primer having a cleavage domain positioned 5′ of ablocking group and 3′ of a position of variation, the blocking grouplinked at or near the end of the 3′-end of the oligonucleotide primerwherein the blocking group prevents primer extension and/or inhibits theprimer from serving as a template for DNA synthesis; (ii) a samplenucleic acid that may or may not have the target sequence, and where thetarget sequence may or may not have the variation; (iii) a cleavingenzyme; and (iv) a polymerase; (b) hybridizing the primer to the targetDNA sequence to form a double-stranded substrate; (c) cleaving thehybridized primer, if the primer is complementary at the variation, withthe cleaving enzyme at a point within or adjacent to the cleavage domainto remove the blocking group from the primer; and (d) extending theprimer with the polymerase.
 14. The method of claim 13, wherein thetarget DNA sequence has been treated with a CRISPR enzyme.
 15. Themethod of claim 13, wherein the target DNA sequence has been treatedwith a Cas9 or Cpf1 enzyme.
 16. The method of claim 13, wherein thecleaving enzyme is a hot start cleaving enzyme which is thermostable andhas reduced activity at lower temperatures.
 17. The method of claim 13,wherein the cleaving enzyme is an RNase H2 enzyme.
 18. The method ofclaim 17, wherein the cleaving enzyme is Pyrococcus abyssi RNase H2enzyme.
 19. The method of claim 13, wherein the cleaving enzyme is achemically modified hot start cleaving enzyme which is thermostable andhas reduced activity at lower temperatures.
 20. The method of claim 19,wherein the hot start cleaving enzyme is a chemically modifiedPyrococcus abyssi RNase H2.
 21. The method of claim 13, wherein thecleaving enzyme is a hot start cleaving enzyme that is reversiblyinactivated through interaction with an antibody at lower temperatures.22. The method of claim 13, wherein the cleavage domain comprises atleast one RNA base, and the cleaving enzyme cleaves between the positioncomplementary to the variation and the RNA base.
 23. The method of claim22, wherein the cleavage domain comprises at least one RNA base located3′ of the position of variation, and comprises one DNA base between theposition of variation and the RNA base.
 24. The method of claim 13,wherein the cleavage domain comprises one or more 2′-modifiednucleosides, and the cleaving enzyme cleaves between the positioncomplementary to the variation and the one or more modified nucleosides.25. The method of claim 24, wherein the one or more modified nucleosidesare 2′-fluoronucleosides.
 26. The method of claim 13, wherein thepolymerase is a high-discrimination polymerase.
 27. The method of claim13, wherein the polymerase is a mutant H784Q Taq polymerase.
 28. Themethod of claim 27, wherein the mutant H784Q Taq polymerase isreversibly inactivated via chemical, aptamer, or antibody modification.29. The method of claim 13, wherein the primer contains a 5′ tailsequence that comprises a universal primer sequence and optionally auniversal probe sequence, wherein the tail is non-complementary to thetarget DNA sequence.
 30. The method of claim 1, further comprising (e)detection of an internal control gene not targeted by the gene editingenzyme; and (f) normalization of the results of steps (a)-(d) to theresults of step (e).
 31. The method of claim 30, wherein the internalcontrol gene not targeted by the gene editing enzyme is the RNase Pgene.
 32. The method of claim 30, wherein the reaction mixture furthercomprises a control oligonucleotide primer specific for the internalcontrol gene not targeted by the gene editing enzyme, wherein thecontrol oligonucleotide primer comprises a cleavage domain positioned 5′of a blocking group and 3′ of a position of variation, the blockinggroup linked at or near the end of the 3′-end of the oligonucleotideprimer wherein the blocking group prevents primer extension and/orinhibits the primer from serving as a template for DNA synthesis. 33.The method of claim 30, wherein the internal control gene not targetedby the gene editing enzyme is detected using a three-oligonucleotide 5′nuclease assay.
 34. A method of target enrichment comprising: (a)providing a reaction mixture comprising: (i) a first oligonucleotideprimer having a tail domain that is not complementary to a targetsequence, the tail domain comprising a first universal primer sequence;a cleavage domain positioned 5′ of a blocking group and 3′ of a positionof variation, the blocking group linked at or near the end of the 3′-endof the first oligonucleotide primer wherein the blocking group preventsprimer extension and/or inhibits the first primer from serving as atemplate for DNA synthesis; (ii) a sample nucleic acid that has beentreated with a gene editing enzyme, which may or may not have the targetsequence; (iii) a cleaving enzyme; and (iv) a polymerase; (b)hybridizing the first primer to the target DNA sequence to form adouble-stranded substrate; (c) cleaving the hybridized first primer, ifthe first primer is complementary to the target, with the cleavingenzyme at a point within or adjacent to the cleavage domain to removethe blocking group from the first primer; and (d) extending the firstprimer with the polymerase.
 35. The method of claim 34, wherein thetarget DNA sequence is a sample that has been treated with a CRISPRenzyme.
 36. The method of claim 34, wherein the target DNA sequence is asample that has been treated with a Cas9 or Cpf1 enzyme.
 37. The methodof claim 34, the method further comprising a second primer in reverseorientation to support priming and extension of the first primerextension product.
 38. The method of claim 37, wherein the second primerfurther comprises a tail domain comprising a second universal primersequence.
 39. The method of claim 38, wherein steps (b)-(d) areperformed 1-10 times.
 40. The method of claim 39, further comprisingremoving unextended primers from the reaction and hybridizing universalprimers to the extension product to form a second extension product. 41.The method of claim 40, wherein the universal primers further comprisetailed sequences for addition of adapter sequences to the secondextension product.
 42. The method of claim 41, wherein sequencing isperformed on the second extension product to determine the sequence ofthe target.
 43. The method of claim 34, wherein the cleaving enzyme is ahot start cleaving enzyme which is thermostable and has reduced activityat lower temperatures.
 44. The method of claim 43, wherein the cleavingenzyme is an RNase H2 enzyme.
 45. The method of claim 44, wherein thecleaving enzyme is Pyrococcus abyssi RNase H2 enzyme.
 46. The method ofclaim 34, wherein the cleaving enzyme is a chemically modified hot startcleaving enzyme which is thermostable and has reduced activity at lowertemperatures.
 47. The method of claim 34, wherein the hot start cleavingenzyme is a chemically modified Pyrococcus abyssi RNase H2.
 48. Themethod of claim 34, wherein the cleaving enzyme is a hot start cleavingenzyme that is reversibly inactivated through interaction with anantibody at lower temperatures which is thermostable and has reducedactivity at lower temperatures.
 49. The method of claim 34, wherein thecleavage domain comprises at least one RNA base, and the cleaving enzymecleaves between the position complementary to the variation and the RNAbase.
 50. The method of claim 49, wherein the cleavage domain comprisesat least one RNA base located 3′ of the position of variation, andcomprises one DNA base between the position of variation and the RNAbase.
 51. The method of claim 34, wherein the cleavage domain comprisesone or more 2′-modified nucleosides, and the cleaving enzyme cleavesbetween the position complementary to the variation and the one or moremodified nucleosides.
 52. The method of claim 51, wherein the one ormore modified nucleosides are 2′-fluoronucleosides.
 53. The method ofclaim 34, wherein the polymerase is a high-discrimination polymerase.54. The method of claim 34, wherein the polymerase is a mutant H784Q Taqpolymerase.
 55. The method of claim 54, wherein the mutant H784Q Taqpolymerase is reversibly inactivated via chemical, aptamer or antibodymodification.